Non-cytotoxic protein conjugates

ABSTRACT

The present invention is directed to non-cytotoxic protein conjugates for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell. The protein conjugates comprise: (i) a Targeting Moiety (TM), wherein the TM is an agonist of a receptor present on a nociceptive sensory afferent cell, and wherein the receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell; (ii) a non-cytotoxic protease or a fragment thereof, wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of the nociceptive sensory afferent cell; and (iii) a Translocation Domain, wherein the Translocation Domain translocates the protease or protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell. Nucleic acid sequences encoding the protein conjugates, methods of preparing same and uses thereof are also described.

This invention relates to a non-cytotoxic protein conjugate, and to theuse of said conjugate for treating pain.

Toxins may be generally divided into two groups according to the type ofeffect that they have on a target cell. In more detail, the first groupof toxins kill their natural target cells, and are therefore known ascytotoxic toxin molecules. This group of toxins is exemplified interalia by plant toxins such as ricin, and abrin, and by bacterial toxinssuch as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxinstypically kill their target cells by inhibiting the cellular process ofprotein synthesis.

In contrast, the second group of toxins, which are known asnon-cytotoxic toxins, do not (as their name confirms) kill their naturaltarget cells. Non-cytotoxic toxins have attracted much less commercialinterest than have their cytotoxic counterparts, and exert their effectson a target cell by inhibiting cellular processes other than proteinsynthesis. As with their cytotoxic counterparts, non-cytotoxic toxinsare produced from a variety of sources such as plants, and bacteria.Bacterial non-cytotoxic toxins are now described in more detail.

Clostridial neurotoxins are proteins that typically have a molecularmass of the order of 150 kDa. They are produced by various species ofbacteria, especially of the genus Clostridium, most importantly C.tetani and several strains of C. botulinum, C. butyricum and C.argentinense. There are at present eight different classes of theclostridial neurotoxin, namely: tetanus toxin, and botulinum neurotoxinin its serotypes A, B, C₁, D, E, F and G, and they all share similarstructures and modes of action.

Clostridial neurotoxins represent a major group of non-cytotoxic toxinmolecules, and are synthesised by the host bacterium as singlepolypeptides that are modified post-translationally by a proteolyticcleavage event to form two polypeptide chains joined together by adisulphide bond. The two chains are termed the heavy chain (H-chain),which has a molecular mass of approximately 100 kDa, and the light chain(L-chain), which has a molecular mass of approximately 50 kDa.

L-chains possess a protease function (zinc-dependent endopeptidaseactivity) and exhibit high substrate specificity for vesicle and/orplasma membrane associated proteins involved in the exocytic process.L-chains from different clostridial species or serotypes may hydrolysedifferent but specific peptide bonds in one of three substrate proteins,namely synaptobrevin, syntaxin or SNAP-25. These substrates areimportant components of the neurosecretory machinery.

Non-cytotoxic toxins are also produced by other bacteria, such as fromthe genus Neisseria, most importantly from the species N. gonorrhoeae.For example, Neisseria sp. produces the non-cytotoxic toxin IgA protease(see WO99/58571).

It has been well documented in the art that toxin molecules may bere-targeted to a cell that is not the toxin's natural target cell. Whenso re-targeted, the modified toxin is capable of binding to a desiredtarget cell and, following subsequent translocation into the cytosol, iscapable of exerting its effect on the target cell. Said re-targeting isachieved by replacing the natural Targeting Moiety (TM) of the toxinwith a different TM. In this regard, the TM is selected so that it willbind to a desired target cell, and allow subsequent passage of themodified toxin into an endosome within the target cell. The modifiedtoxin also comprises a translocation domain to enable entry of thenon-cytotoxic protease into the cell cytosol. The translocation domaincan be the natural translocation domain of the toxin or it can be adifferent translocation domain obtained from a microbial protein withtranslocation activity.

For example, in the context of non-cytotoxic toxin molecules, it hasbeen well documented that a clostridial neurotoxin may be re-targeted byincorporation of a Targeting Moiety (TM), which is not the natural TM ofa clostridial neurotoxin. The described chemical conjugation andrecombinant methodologies are now regarded as conventional, andreference is made to Hermanson, G. T. (1996), Bioconjugate techniques,Academic Press, and to Wong, S. S. (1991), Chemistry of proteinconjugation and cross-linking, CRC Press.

For example, WO94/21300 describes modified clostridial neurotoxinmolecules that are capable of regulating Integral Membrane Protein (IMP)density present at the cell surface of the target cell. The modifiedneurotoxin molecules are thus capable of controlling cell activity (e.g.glucose uptake) of the target cell. WO96/33273 and WO99/17806 describemodified clostridial neurotoxin molecules that target peripheral sensoryafferents. The modified neurotoxin molecules are thus capable ofdemonstrating an analgesic effect. WO00/10598 describes the preparationof modified clostridial neurotoxin molecules that target mucushypersecreting cells (or neuronal cells controlling said mucushypersecreting cells), which modified neurotoxins are capable ofinhibiting hypersecretion from said cells. WO01/21213 describes modifiedclostridial neurotoxin molecules that target a wide range of differenttypes of non-neuronal target cells. The modified molecules are thuscapable of preventing secretion from the target cells. Additionalpublications in the technical field of re-targeted toxin moleculesinclude: WO00/62814; WO00/04926; U.S. Pat. No. 5,773,586; WO93/15766;WO00/61192; and WO99/58571.

Thus, from the above-described publications, it will be appreciated thatthe basic concept of re-targeting a non-cytotoxic protease to a desiredtarget cell, by selecting a TM that has a corresponding receptor presenton the target cell, has been well documented.

However, different receptors present on a target cell of interestdemonstrate different binding affinities for different TMs. This may bea particular problem with pain-sensing cells, which possess a wide rangeof receptor types having different binding affinities for different TMs.Thus, a re-targeted conjugate comprising a particular TM (that binds toa receptor on a pain-sensing cell) may demonstrate a low bindingaffinity for a pain-sensing target cell, which is undesirable.

There is therefore a need to develop modified non-cytotoxic conjugatesthat address one or more of the above problems. Of particular interestis the development of an improved conjugate for use in treating pain.

The present invention seeks to address one or more of the above problemsby using as the conjugate's Targeting Moiety (TM) an “agonist” of areceptor that is present on the pain-sensing target cell of interest. Inpreferred embodiments, the pain-sensing target cell is a nociceptivesensory afferent, more preferably a primary nociceptive sensoryafferent. In particularly preferred embodiments, the TM is an agonist ofthe opioid-like receptor-1 (ORL₁) receptor.

Accordingly, in a first aspect, the present invention provides anon-cytotoxic conjugate for inhibition or reduction of exocytic fusionin a nociceptive sensory is afferent cell, comprising:

-   -   (i) a Targeting Moiety (TM),        -   wherein said TM is an agonist of a receptor present on said            nociceptive sensory afferent cell, and wherein said receptor            undergoes endocytosis to be incorporated into an endosome            within the nociceptive sensory afferent cell;    -   (ii) a non-cytotoxic protease or a fragment thereof,        -   wherein the protease or protease fragment is capable of            cleaving a protein of the exocytic fusion apparatus of said            nociceptive sensory afferent cell; and    -   (iii) a Translocation Domain,        -   wherein the Translocation Domain translocates the protease            or protease fragment from within the endosome, across the            endosomal membrane, and into the cytosol of the nociceptive            sensory afferent cell.

The use of an “agonist”, which would normally stimulate a biologicalprocess, particularly exocytosis (for example, an increase in cellularsecretion, or an up-regulation in membrane protein expression), is anexciting development in the technical field of re-targeted toxins.Furthermore, it is particularly surprising that an agonist may beemployed in a therapeutic composition to achieve a reduction orinhibition of a biological process that the agonist would normallystimulate.

The agonist-containing conjugates of the present invention represent adistinct sub-set of toxin conjugates. In more detail, the conjugates ofthe present invention comprise TMs that have been selected on the basisof specific agonist properties rather than on the simple basis that theyhave a corresponding receptor on a pain-sensing target cell of interest.

Conventionally, an agonist has been considered any molecule that caneither increase or decrease activities within a cell, namely anymolecule that simply causes an alteration of cell activity. For example,the conventional meaning of an agonist would include: a chemicalsubstance capable of combining with a receptor on a cell and initiatinga reaction or activity, or a drug that induces an active response byactivating receptors, whether the response is an increase or decrease incellular activity.

However, for the purposes of this invention, an agonist is morespecifically defined as a molecule that is capable of stimulating theprocess of exocytic fusion in a pain-sensing target cell, which processis susceptible to inhibition by a protease (or fragment thereof) capableof cleaving a protein of the exocytic fusion apparatus in said targetcell.

Accordingly, the particular agonist definition of the present inventionwould exclude many molecules that would be conventionally considered asagonists. For example, nerve growth factor (NGF) is an agonist inrespect of its ability to promote neuronal differentiation via bindingto a TrkA receptor. However, NGF is not an agonist when assessed by theabove criteria because it is not a principal inducer of exocytic fusion.In addition, the process that NGF stimulates (i.e. cell differentiation)is not susceptible to inhibition by the protease activity of anon-cytotoxic toxin molecule.

In use, an agonist-containing conjugate of the present invention doesnot deactivate an agonist receptor on a pain-sensing target cell, butrather the protease activity of the conjugate serves to negate theagonist-mediated response.

Furthermore, once delivered to the cytosol of the pain-sensing targetcell, the protease component of a conjugate of the present inventioninhibits or blocks the action of all subsequent agonists capable ofcausing the same effect (i.e. increased exocytic fusion) in the sametarget cell. This is advantageous and means that the conjugates of thepresent invention have application in situations where multiple agonistsmay be responsible for causing the sensation of pain. Thus, whendesigning a conjugate of the present invention, the TM that is selectedfor delivery need not necessarily be the principal agonist involved incausing the sensation of pain.

Agonist-mediated delivery according to the present invention providesthe following significant advantage over previous non-cytotoxicprotease-containing therapeutics: use of an agonist may conferpreferential binding and/or internalisation properties on the conjugate.This, in turn, may result in more efficient delivery of the proteasecomponent to a pain-sensing target cell.

In addition, use of an agonist as a TM is self-limiting with respect toside-effects. In more detail, binding of an agonist to a pain-sensingtarget cell increases exocytic fusion, which may exacerbate thesensation of pain. However, the exocytic process that is stimulated byagonist binding is subsequently reduced or inhibited by the proteasecomponent of the conjugate.

In preferred embodiments of the invention, the TM is an agonist of theORL₁ receptor. The ORL₁ receptor is present on pain-sensing cells in thebody.

The ORL₁ receptor is a member of the G-protein-coupled class ofreceptors, and has a seven transmembrane domain structure. Theproperties of the ORL₁ receptor are discussed in detail in Mogil &Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pages381-415.

Throughout this specification, reference to the “ORL₁ receptor” embracesall members of the ORL₁ receptor family. Members of the ORL₁ receptorfamily typically have a seven transmembrane domain structure, and arecoupled to G-proteins of the G_(i) and G₀ families. A method fordetermining the G-protein-stimulating activity of ligands of the ORL₁receptor is given in Example 17. A method for measuring reduction incellular cAMP levels following ORL₁ activation is given in Example 16. Afurther characteristic of members of the ORL₁ receptor family is thatthey are typically able to bind nociceptin (the natural ligand of ORL₁).As an example, all alternative splice variants of the ORL₁ receptor, aremembers of the ORL₁ receptor family.

The conjugates of the present invention generally demonstrate a reducedbinding affinity (in the region of up to 100-fold) for nociceptivesensory afferent target cells when compared with the corresponding‘free’ TM. However, despite this observation, the conjugates of thepresent invention surprisingly demonstrate good efficacy. This can beattributed to two principal features. First, the non-cytotoxic proteasecomponent is catalytic—thus, the therapeutic effect of a few suchmolecules is rapidly amplified. Secondly, the receptors present on thenociceptive sensory afferents need only act as a gateway for entry ofthe therapeutic, and need not necessarily be stimulated to a levelrequired in order to achieve a ligand-receptor mediated pharmacologicalresponse. Accordingly, the conjugates of the present invention may beadministered at a dosage that is much lower that would be employed forother types of analgesic molecules such as NSAIDS, morphine, andgabapentin. The latter molecules are typically administered at highmicrogram to milligram (even up to hundreds of milligram) quantities,whereas the conjugates of the present invention may be administered atmuch lower dosages, typically at least 10-fold lower, and more typicallyat 100-fold lower.

In a particularly preferred embodiment of the invention, the TM of theconjugate is nociceptin—the natural ligand for the ORL₁ receptor.Nociceptin targets the ORL₁ receptor with high affinity.

Examples of other preferred TMs include:

SEQ  Code Sequence Ref. ID NO: Nociceptin FGGFTGARKSARKLANQ [1]  1, 21-17 Nociceptin FGGFTGARKSA [1]  3, 4 1-11 Nociceptin FGGFTGARKYA [1] 5, 6 [Y10]1-11 Nociceptin FGGFTGARKSY [1]  7, 8 [Y11]1-11 NociceptinFGGFTGARKSARKYANQ [1]   9, 10 [Y14]1-17 Nociceptin FGGFTGARKSARK [2] 11, 12 1-13 Nociceptin FGGFTGARKSARKRKNQ [3, 4]  13, 14 [R14K15]1-17 (also known as “variant” nociceptin) Nociceptin FGGFTGARKSARK-NH₂[5] 12 [[-]] 1-13-NH₂ Nociceptin (pNO₂)FGGFTGARKSAR [5]  2 [[-]]Phe (p-NO₂) KLANQ 1-17 Lofentanil Non-peptide [5] — agonists EtorphineNon-peptide [5] — agonists Peptide Peptide agonists [6] — agonistfrom combinatorial library approach [1] Mogil & Pasternak, 2001,Pharmacol. Rev., 53, 381-415 [2] Maile et al., 2003, Neurosci. Lett.,350, 190-192 [3] Rizzi et al., 2002, J. Pharmacol. Exp. Therap., 300,57-63 [4] Okada et al., 2000, Biochem. Biophys. Res. Commun., 278,493-498 [5] Zaveri, 2003, Life Sci., 73, 663-678. [6] Dooley et al.,1997, J Pharmacol Exp Ther. 283(2), 735-41.

The TM preferably comprises a maximum of 50 amino acid residues, morepreferably a maximum of 40 amino acid residues, particularly preferablya maximum of 30 amino acid residues, and most preferably a maximum of 20amino acid residues. For example, nociceptin is a 17 amino acid residuepeptide.

The above-identified “variant” TM demonstrates particularly good bindingaffinity (when compared with natural nociceptin) for nociceptive sensoryafferents. Generally speaking, a TM-containing conjugate willdemonstrate an approximate 100-fold reduction in binding abilityvis-à-vis the TM per se. The above-mentioned “variant” TM per sedemonstrates an approximate 3- to 10-fold increase in binding abilityfor a nociceptive sensory afferent vis-à-vis natural nociceptin. Thus, a“variant” TM-containing fusion might be expected to demonstrate anapproximate 10-fold reduction in binding ability for a nociceptivesensory afferent vis-à-vis ‘free’ nociceptin. However, the presentinventors have demonstrated that conjugates comprising said “variant” TMdemonstrate a binding ability that (most surprisingly) closely mirrorsthat of ‘free’ nociceptin—see FIG. 17.

In the context of the present invention, the term agonist of the ORL₁receptor (such as nociceptin, or any one of the peptides listed in thetable above) embraces molecules having at least 70%, preferably at least80%, more preferably at least 90%, and most preferably at least 95%homology with said agonist. The agonist homologues retain the agonistproperties of nociceptin at the ORL₁ receptor, which may be tested usingthe methods provided in Example 10.

The invention also encompasses fragments, variants, and derivatives ofany one of the TMs described above. These fragments, variants, andderivatives will substantially retain the properties that are ascribedto said TMs.

The agonist properties of a TM can be confirmed using the methodsdescribed in Example 1. These methods are based on previous experiments(see Inoue et al. (1998) Proc. Natl. Acad. Sci., 95, 10949-10953), whichconfirm that the natural agonist of the ORL₁ receptor, nociceptin,causes the induction of substance P release from nociceptive primaryafferent neurons. This is supported by the facts that:

-   -   the nociceptin-induced responses are abolished by specific NK1        receptor (the substance P receptor) antagonists; and    -   pre-treatment of the cells with capsaicin (which depletes        substance P from small diameter primary afferent neurons)        attenuates the nociceptin-induced responses.

Similarly, Inoue et al. confirm that an intraplantar injection ofbotulinum neurotoxin type A abolishes the nociceptin-induced responses.Since it is known that BoNT inhibits the release of substance P fromprimary afferent neurons (Welch et al., (2000), Toxicon, 38, 245-258),this confirms the link between nociceptin-ORL₁ interaction andsubsequent release of substance P.

Thus, a TM can be said to have agonist activity at the ORL₁ receptor ifthe TM causes an induction in the release of substance P from anociceptive sensory afferent neuron (see Example 1).

In another embodiment, opioids represent a preferred group of TMs of thepresent invention. Within this family of peptides is includedenkephalins (met and leu), endomorphins 1 and 2, β-endorphin anddynorphin. Opioid peptides are frequently used in the clinic to modifythe activity to nociceptors, and other cells involved in the painresponse. As exemplified by the three-step World Health OrganisationAnalgesic Ladder, opioids have entry points into the pharmacologicaltreatment of chronic cancer and non-cancer pain at all three stages,underlining their importance to the treatment of pain. Reference toopioids embraces fragments, variants and derivatives thereof, whichretain the ability to bind to nociceptive sensory afferents. Theprotease of the present invention embraces all naturally-occurringnon-cytotoxic proteases that are capable of cleaving one or moreproteins of the exocytic fusion apparatus in eukaryotic cells.

The protease of the present invention is preferably a bacterialprotease.

More preferably, the bacterial protease is selected from the generaClostridium or Neisseria (e.g. a clostridial L-chain, or a neisserialIgA protease preferably from N. gonorrhoeae).

The present invention also embraces modified non-cytotoxic proteases,which include amino acid sequences that do not occur in nature and/orsynthetic amino acid residues, so long as the modified proteases stilldemonstrate the above-mentioned protease activity.

The protease of the present invention preferably demonstrates a serineor metalloprotease activity (e.g. endopeptidase activity). The proteaseis preferably specific for a SNARE protein (e.g. SNAP-25,synaptobrevin/VAMP, or syntaxin).

Particular mention is made to the protease domains of neurotoxins, forexample the protease domains of bacterial neurotoxins. Thus, the presentinvention embraces the use of neurotoxin domains, which occur in nature,as well as recombinantly prepared versions of said naturally-occurringneurotoxins.

Exemplary neurotoxins are produced by clostridia, and the termclostridial neurotoxin embraces neurotoxins produced by C. tetani(TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closelyrelated BoNT-like neurotoxins produced by C. baratii and C. butyricum.The above-mentioned abbreviations are used throughout the presentspecification. For example, the nomenclature BoNT/A denotes the sourceof neurotoxin as BoNT (serotype A). Corresponding nomenclature appliesto other BoNT serotypes.

The term L-chain fragment means a component of the L-chain of aneurotoxin, which fragment demonstrates a metalloprotease activity andis capable of proteolytically cleaving a vesicle and/or plasma membraneassociated protein involved in cellular exocytosis.

A Translocation Domain is a molecule that enables translocation of aprotease (or fragment thereof) into a pain-sensing target cell such thata functional expression of protease activity occurs within the cytosolof the target cell. Whether any molecule (e.g. a protein or peptide)possesses the requisite translocation function of the present inventionmay be confirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employingliposomes, which are challenged with a test molecule. Presence of therequisite translocation function is confirmed by release from theliposomes of K⁺ and/or labelled NAD, which may be readily monitored (seeShone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180).

A further example is provided by Blaustein R. (1987), which describes asimple in vitro assay employing planar phospholipid bilayer membranes.The membranes are challenged with a test molecule and the requisitetranslocation function is confirmed by an increase in conductance acrosssaid membranes (see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp.115-120).

Additional methodology to enable assessment of membrane fusion and thusidentification of Translocation Domains suitable for use in the presentinvention are provided by Methods in Enzymology, Vols. 220 and 221,Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The Translocation Domain is preferably capable of formation ofion-permeable pores in lipid membranes under conditions of low pH.Preferably, it has been found to use only those portions of the proteinmolecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial proteinsource, in particular from a bacterial or viral protein source. Hence,in one embodiment, the Translocation Domain is a translocating domain ofan enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin moleculesare capable of forming such pores. It is also known that certaintranslocation domains of virally expressed membrane fusion proteins arecapable of forming such pores. Such domains may be employed in thepresent invention.

The Translocation Domain may be of a clostridial origin, namely theH_(N) domain (or a functional component thereof). H_(N) means a portionor fragment of the H-chain of a clostridial neurotoxin approximatelyequivalent to the amino-terminal half of the H-chain, or the domaincorresponding to that fragment in the intact H-chain. Examples ofsuitable clostridial Translocation Domains include:

-   -   Botulinum type A neurotoxin—amino acid residues (449-871)    -   Botulinum type B neurotoxin—amino acid residues (441-858)    -   Botulinum type C neurotoxin—amino acid residues (442-866)    -   Botulinum type D neurotoxin—amino acid residues (446-862)    -   Botulinum type E neurotoxin—amino acid residues (423-845)    -   Botulinum type F neurotoxin—amino acid residues (440-864)    -   Botulinum type G neurotoxin—amino acid residues (442-863)    -   Tetanus neurotoxin—amino acid residues (458-879)

For further details on the genetic basis of toxin production inClostridium botulinum and C. tetani, we refer to Henderson et al. (1997)in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_(N) embraces naturally-occurring neurotoxin H_(N) portions,and modified H_(N) portions having amino acid sequences that do notoccur in nature and/or synthetic amino acid residues, so long as themodified H_(N) portions still demonstrate the above-mentionedtranslocation function.

Alternatively, the Translocation Domain may be of a non-clostridialorigin (see table below). Examples of non-clostridial TranslocationDomain origins include, but are not restricted to, the translocationdomain of diphtheria toxin [O'Keefe et al., Proc. Natl. Acad. Sci. USA(1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269,22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], the translocation domain of Pseudomonas exotoxin type A [Prioret al. Biochemistry (1992) 31, 3555-3559], the translocation domains ofanthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93,8437-8442], a variety of fusogenic or hydrophobic peptides oftranslocating function [Plank et al. J. Biol. Chem. (1994) 269,12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938], andamphiphilic peptides [Murata et al (1992) Biochem., 31, pp. 1986-1992].The Translocation Domain may mirror the Translocation Domain present ina naturally-occurring protein, or may include amino acid variations solong as the variations do not destroy the translocating ability of theTranslocation Domain.

Particular examples of viral Translocation Domains suitable for use inthe present invention include certain translocating domains of virallyexpressed membrane fusion proteins. For example, Wagner et al. (1992)and Murata et al. (1992) describe the translocation (i.e. membranefusion and vesiculation) function of a number of fusogenic andamphiphilic peptides derived from the N-terminal region of influenzavirus haemagglutinin. Other virally expressed membrane fusion proteinsknown to have the desired translocating activity are a translocatingdomain of a fusogenic peptide of Semliki Forest Virus (SFV), atranslocating domain of vesicular stomatitis virus (VSV) glycoprotein G,a translocating domain of SER virus F protein and a translocating domainof Foamy virus envelope glycoprotein. Virally encoded “spike proteins”have particular application in the context of the present invention, forexample, the E1 protein of SFV and the G protein of VSV.

Use of the Translocation Domains (listed below) includes use of sequencevariants thereof. A variant may comprise one or more conservativenucleic acid substitutions and/or nucleic acid deletions or insertions,with the proviso that the variant possesses the requisite translocatingfunction. A variant may also comprise one or more amino acidsubstitutions and/or amino acid deletions or insertions, so long as thevariant possesses the requisite translocating function.

Translocation Amino acid Domain source residues References Diphtheria194-380 Silverman et al., 1994, toxin J. Biol. Chem. 269, 22524-22532London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of405-613 Prior et al., 1992, pseudomonas Biochemistry 31, 3555-3559exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virusGLFGAIAGFIENGWE Plank et al., 1994, haemagglutinin GMIDGWYG J. Biol.Chem. 269, (SEQ ID NO: 101), 12918-12924 and Wagner et al., 1992,Variants thereof PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry31, 1986-1992 Semliki Forest Translocation domain Kielian et al., 1996,virus fusogenic J Cell Biol. 134(4), 863-872 protein Vesicular 118-139Yao et al., 2003, Stomatitis Virology 310(2), 319-332 virus glycoproteinG SER virus F Translocation domain Seth et al., 2003, protein J Virol77(11) 6520-6527 Foamy virus Translocation domain Picard-Maureau et al.,2003, envelope J Virol. 77(8), 4722-4730 glycoprotein

Once a potential receptor agonist (e.g. an ORL1 agonist) has beenidentified, one or more of the following optional steps may be carriedout:

-   -   (A) confirming that the putative agonist molecule or agonist is        capable of being combined with a non-cytotoxic protease (or a        fragment thereof) and optionally a Translocation Domain to form        a conjugate of the present invention; and/or    -   (B) confirming that said putative agonist molecule or agonist        binds to the receptor on the pain-sensing target cell, which        receptor is susceptible to receptor-mediated endocytosis; and/or    -   (C) confirming that said putative agonist molecule or agonist is        able to deliver a non-cytotoxic protease (or fragment thereof)        into the cytosol of a pain-sensing target cell.

The above steps (A)-(C) may be confirmed by routine tests that would bereadily available to a skilled person.

For example, step (A) may be performed by a simple chemical conjugationexperiment using conventional conjugation reagents and/or linkermolecules, followed by native polyacrylamide gel electrophoresis toconfirm that a conjugate of the present invention is formed that has theanticipated molecular weight. The conjugate components are typicallylinked together (optionally via linker molecules) by covalent bonds.

For example, step (B) may be performed by any one of a range ofmethodologies for assessment of binding of a ligand. Standard text, forexample “Receptor-Ligand Interactions. A Practical Approach. Ed. E. C.Hulme, IRL Press, 1992” are available that describe such approaches indetail. In brief, the agonist or putative agonist molecule is labelled(for example, with 125-iodine) and applied to a cell preparation invitro in the presence of an excess of unlabelled agonist. The purpose ofthe unlabelled material is to saturate any non-specific binding sites.The agonist is incubated with the cell preparation for sufficient timeto achieve equilibrium, and the amount of label bound to the cellsassessed by measuring cell associated radioactivity, for example byscintillation or gamma counting.

A further example involves gold-labelling of the agonist (or putativeagonist), followed by the use of electron microscopy to monitor thecellular transport progress of the labelled agonist [see the basicmethodology described by Rabinowitz S. (1992); J. Cell. Biol. 116(1):pp. 95-112; and that described by van Deurs (1986); J. Cell. Biol. 102:pp. 37-47].

For example, step (C) may be performed by contacting the conjugateprepared in step (A) with a suitable target cell and assessing cleavageof the substrate. This is performed by extraction of the SNARE proteins,followed by Western blotting of SDS-PAGE-separated samples. Cleavage ofsubstrate is indicative of delivery of the protease into the targetcell. In this regard, cleavage may be monitored by disappearance ofsubstrate and/or appearance of cleavage product. A particularly usefulantibody that selectively binds to the cleaved substrate product isdescribed in WO95/33850.

Preparation of a conjugate according to the present invention is nowdiscussed.

It is known in the art that the H_(C) portion of a neurotoxin moleculecan be removed from the other portion of the H-chain, known as H_(N),such that the H_(N) fragment remains disulphide linked to the L-chain ofthe neurotoxin providing a fragment known as LH_(N). Thus, in oneembodiment of the present invention the LH_(N) fragment of a neurotoxinis covalently linked, using linkages which may include one or morespacer regions, to a TM.

In another embodiment of the invention, the H_(C) domain of a neurotoxinis mutated, blocked or modified, e.g. by chemical modification, toreduce or preferably incapacitate its ability to bind the neurotoxin toreceptors at the neuromuscular junction. This modified neurotoxin isthen covalently linked, using linkages which may include one or morespacer regions, to a TM.

In another embodiment of the invention, the H-chain of a neurotoxin, inwhich the H_(C) domain is mutated, blocked or modified, e.g. by chemicalmodification, to reduce or preferably incapacitate its native bindingability, is combined with the L-chain of a different neurotoxin, oranother protease capable of cleaving a protein of the exocytic fusionapparatus (e.g. IgA protease of N. gonorrhoeae). This hybrid, modifiedneurotoxin is then covalently linked, using linkages which may includeone or more spacer regions, to a TM.

In another embodiment of the invention, the H_(N) domain of a neurotoxinis combined with the L-chain of a different neurotoxin, or anotherprotease capable of cleaving a protein of the exocytic fusion apparatus(e.g. IgA protease of N. gonorrhoeae). This hybrid is then covalentlylinked, using linkages which may include one or more spacer regions, toa TM.

In another embodiment of the invention, the protease (for example theL-chain component of a neurotoxin) is covalently linked, using linkagesthat may include one or more spacer regions, to a TM that can alsoeffect the internalisation of the protease into the cytoplasm of therelevant target cell(s).

In another embodiment of the invention, the protease (for example theL-chain component of a neurotoxin) is covalently linked, using linkageswhich may include one or more spacer regions, to a translocation domainto effect transport of the protease fragment into the cytosol.

In use, the domains of a conjugate according to the present inventionare associated with each other. In one embodiment, two or more of thedomains may be joined together either directly (e.g. by a covalentlinkage), or via a linker molecule.

A variety of different linker/spacer molecules may be employed in any ofthe fusion proteins of the present invention. Examples of such spacermolecules include those illustrated in FIGS. 31 and 32. Particularmention here is made to GS15, GS20, GS25, and Hx27—see FIGS. 31 and 32.

The present inventors have unexpectedly found that non-cytotoxicprotease-TM conjugates (eg. CPNv/A) may demonstrate an improved bindingactivity for nociceptive sensory afferents when the size of the spaceris selected so that (in use) the TM (preferably the C-terminus thereof)and the translocation domain (preferably the N-terminus thereof) areseparated from one another by 40-105 angstroms, preferably by 50-100angstroms, and more preferably by 50-90 angstroms. In anotherembodiment, the preferred spacers have an amino acid sequence of 11-29amino acid residues, preferably 15-27 amino acid residues, and morepreferably 20-27 amino acid residues. Suitable spacers may be routinelyidentified and obtained according to Crasto, C. J. and Feng, J. A.(2000) May, 13(5), pp. 309-312—see also the website:fccc./edu/research/labs/feng/limker.html

Conjugation techniques suitable for use in the present invention havebeen well documented and are routine for a person skilled in the art.

The methodology involved in coupling two protein molecules (A and B)together is simple, and is achieved through the use of a cross-linkingagent (also known as a chemical coupling agent). For example, moleculesA and B are separately contacted with a cross-linking agent, whichchemically modifies a specific surface group on each of molecules A andB thereby forming derivatised molecules A′ and B′. The modified surfacegroup on molecule A′ is capable of covalently bonding with the modifiedsurface group on molecule B′. Thus, the coupling reaction is completedby mixing together the two protein molecules A′ and B′.

Chemical conjugation is illustrated by reference to the followingembodiments, where P=non-cytotoxic protease component, T=translocationcomponent, and TM=targeting moiety.

In one embodiment, a single chain P-T is prepared, which is thenconjugated to a TM. In another embodiment, a single chain TM-T (or T-TM)is prepared, which is then conjugated to a P. In a further embodiment, asingle chain P-TM (or TM-P) is prepared, which is then conjugated to aT. Another particularly preferred conjugate has the structure P-TM-T(with an optional protease cleavage site between P and TM).

Where the T and P components are prepared as a single chain polypeptide,a protease cleavage site is typically included between said components.Any protease cleavage site may be employed in this regard.

In an alternative embodiment, the three components may be simultaneouslyor sequentially conjugated together. Thus, the conjugation may be a one-or two-step process, and may include one or more different couplingagents.

Chemical coupling agents and cross-linking agents have been commerciallyavailable for many years.

Example 5 of the present invention describes in detail the use of onesuch coupling agent, namely SPDP, to chemically couple two proteinmolecules (nociceptin, and the LH_(N) of botulinum neurotoxin). The twomolecules are separately contacted with SPDP, and then mixed together toallow covalent conjugation.

The conjugate described in Example 6 confirms that another couplingagent, PDPH/EDAC, or Traut's reagent, may be employed as an alternativecoupling agent to SPDP.

SPDP and Traut's reagent are popular and well-documented coupling agentsin the technical field of protein conjugation chemistry and arepresented here simply as two examples of a well known class of compoundsthat may be employed to covalently link together the Targeting Moietycomponent and the clostridial neurotoxin component of the conjugate ofthe present invention. Other suitable agents include SMPB, SMCC(succinimidyl 4-(N-maleimidomethyl)cyclohexan-1-carboxylate), andLC-SPDP.

In more detail, commercially available members of the well-knowncoupling agents may be used for conjugation purposes to produce aconjugate of the invention. Details of such agents can be found in thefollowing publications:

-   -   Hermanson, G. T. (1996), Bioconjugate techniques, Academic        Press;    -   Wong, S. S. (1991), Chemistry of protein conjugation and        cross-linking, CRC Press;    -   Thorpe et al (1987), Cancer Res, 1987, 47, 5924-31. This paper        describes the use of SMBT (sodium S-4-succinimidyloxycarbonyl        -alpha-methyl benzyl thiosulfate) and SMPT        (4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene);        and    -   Peeters et al (1989), J Immunol Methods. 1989, 120, 133-43. This        paper describes the use of 4 coupling reagents, MHS        (succinimidyl 6-(N-maleimido)-n-hexanoate), SMCC (succinimidyl        4-(N -maleimidomethyl)-cyclohexane-1-carboxylate), MBS        (succinimidyl m -maleimidobenzoate), and SPDP.

The conjugates according to the present invention may also be preparedrecombinantly, as detailed in Examples 9 to 12.

In one embodiment, the preparation of a recombinant conjugate involvesarrangement of the coding sequences of a selected TM, a selectednon-cytotoxic protease component, and a translocation component (in anyorder) in a single genetic construct. These coding sequences may bearranged in-frame so that subsequent transcription and translation iscontinuous through both coding sequences and results in a fusionprotein. All constructs would have a 5′ ATG codon to encode anN-terminal methionine, and a C-terminal translational stop codon.

Thus, the recombinant preparation method results in the generation of asingle chain polypeptide. In order to activate this polypeptide, aprotease cleavage site is present between the non-cytotoxic proteasecomponent and the translocation component. Cleavage of this sitegenerates a di-chain polypeptide in which the protease and translocationdomains are linked together by way of a covalent bond, preferably adisulphide bond. In this regard, any protease cleavage site may beemployed.

In the single polypeptide aspect of the present invention, the TM ispreferably either N- or C-terminally located with respect to the fusionprotein. In other words, it is preferred that the TM is not locatedbetween the P and T components of the single polypeptide fusion protein.In a particularly preferred embodiment, the TM is N-terminally locatedwith respect to the fusion protein.

In one embodiment, an L-chain of a clostridial neurotoxin or anotherprotease capable of cleaving a protein of the exocytic fusion apparatus(e.g. an IgA protease), or a fragment/variant thereof, may be expressedrecombinantly as a fusion protein with a TM, which TM can also effectthe internalisation of the L-chain component into the cytoplasm of therelevant target cell(s) responsible for secretion. Alternatively, thefusion protein may further comprise a Translocation Domain. Theexpressed fusion protein may include one or more spacer regions.

By way of example, the following information is required to produce,recombinantly, an agent of the present invention:

-   -   (I) DNA sequence data relating to a selected TM;    -   (II) DNA sequence data relating to the protease component;    -   (III) DNA sequence data relating to the translocation domain;        and    -   (IV) a protocol to permit construction and expression of the        construct comprising (I), (II) and (III).

All of the above basic information (I)-(IV) are either readilyavailable, or are readily determinable by conventional methods. Forexample, both WO98/07864 and WO99/17806 exemplify recombinant technologysuitable for use in the present application.

In addition, methods for the construction and expression of theconstructs of the present invention may employ information from thefollowing references and others:

-   -   Lorberboum-Galski, H., FitzGerald, D., Chaudhary, V., Adhya, S.,        Pastan, I. (1988), Cytotoxic activity of an interleukin        2-Pseudomonas exotoxin chimeric protein produced in Escherichia        coli. Proc. Natl. Acad. Sci. USA, 85(6):1922-6;    -   Murphy, J. R. (1988), Diphtheria-related peptide hormone gene        fusions: a molecular genetic approach to chimeric toxin        development. Cancer Treat. Res.; 37:123-40;    -   Williams, D. P., Parker, K., Bacha, P., Bishai, W., Borowski,        M., Genbauffe, F., Strom, T. B., Murphy, J. R. (1987),        Diphtheria toxin receptor binding domain substitution with        interleukin-2: genetic construction and properties of a        diphtheria toxin-related interleukin-2 fusion protein. Protein        Eng; 1(6):493-8;    -   Arora, N., Williamson, L. C., Leppla, S. H., Halpern, J. L.        (1994), Cytotoxic effects of a chimeric protein consisting of        tetanus toxin light chain and anthrax toxin lethal factor in        non-neuronal cells J. Biol. Chem., 269(42):26165-71;    -   Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., Pastan, I.        (1993), A recombinant immunotoxin containing a        disulphide-stabilized Fv fragment. Proc. Natl. Acad. Sci. USA,        90(16):7538-42; and    -   O'Hare, M., Brown, A. N., Hussain, K., Gebhardt, A., Watson, G.,        Roberts, L. M., Vitetta, E. S., Thorpe, P. E., Lord, J. M.        (1990), Cytotoxicity of a recombinant ricin-A-chain fusion        protein containing a proteolytically-cleavable spacer sequence.        FEBS Lett October 29; 273(1-2):200-4.

Suitable clostridial neurotoxin sequence information relating to L- andLH_(N)-chains may be obtained from, for example, Kurazono, H. (1992) J.Biol. Chem., vol. 267, No. 21, pp. 14721-14729; and Popoff, M. R., andMarvaud, J.-C. (1999) The Comprehensive Sourcebook of Bacterial ProteinToxins, 2nd edition (ed. Alouf, J. E., and Freer, J. H.), AcademicPress, pp. 174-201.

All of the aforementioned publications are hereby incorporated into thepresent specification by reference thereto.

Similarly, suitable TM sequence data are widely available in the art.Alternatively, any necessary sequence data may be obtained by techniqueswhich are well-known to the skilled person.

For example, DNA encoding the TM component may be cloned from a sourceorganism by screening a cDNA library for the correct coding region (forexample by using specific oligonucleotides based on the known sequenceinformation to probe the library), isolating the TM DNA, sequencing thisDNA for confirmation purposes, and then placing the isolated DNA in anappropriate expression vector for expression in the chosen host.

As an alternative to isolation of the sequence from a library, theavailable sequence information may be employed to prepare specificprimers for use in PCR, whereby the coding sequence is then amplifieddirectly from the source material and, by suitable use of primers, maybe cloned directly into an expression vector.

Another alternative method for isolation of the coding sequence is touse the existing sequence information and synthesise a copy, possiblyincorporating alterations, using DNA synthesis technology. For example,DNA sequence data may be generated from existing protein and/or RNAsequence information. Using DNA synthesis technology to do this (and thealternative described above) enables the codon bias of the codingsequence to be modified to be optimal for the chosen expression host.This may give rise to superior expression levels of the fusion protein.

Optimisation of the codon bias for the expression host may be applied tothe DNA sequences encoding the TM and clostridial components of theconstruct. Optimisation of the codon bias is possible by application ofthe protein sequence into freely available DNA/protein databasesoftware, e.g. programs available from Genetics Computer Group, Inc.

Having prepared a conjugate of the invention, it is a matter of routineto confirm that the various domains have retained their specifiedfunction.

Protease function after conjugation may be tested by using, for example,any one of the following routine tests:

SNAP-25 (or synaptobrevin, or syntaxin) may be challenged with aconjugate to be tested, and then analysed by SDS-PAGE peptide separationtechniques. Subsequent detection of peptides (e.g. by silver staining)having molecular weights corresponding to the cleaved products ofSNAP-25 (or other component of the neurosecretory machinery) wouldconfirm the presence of a functional L-chain.

As a further alternative, the conjugate may be tested by assaying forSNAP-25 (or synaptobrevin, or syntaxin) cleavage products viaantibody-specific binding (see WO95/33850). In more detail, a specificantibody is employed for detecting cleavage of SNAP-25. Since theantibody recognises cleaved SNAP-25, but not uncleaved SNAP-25,identification of the cleaved product by the antibody confirms thepresence of L-chain proteolytic function. By way of exemplification,such a method is described in Examples 2 and 3 of WO96/33273.

Translocation component function after conjugation may be tested using,for example, any one of the following routine tests:

Suitable methods are, for example, described by Shone et al. (1987) Eur.J. Biochem. 167, pp. 175-180; and by Blaustein et al. (1987) FEBS 226(1), pp. 115-120.

The Shone et al. method employs artificial liposomes loaded withpotassium phosphate buffer (pH 7.2) and radiolabelled NAD. Release of K+and NAD from the liposomes correlates with a positive result for channelforming activity and hence translocation activity. In this regard, K+release from liposomes may be measured using an electrode and NADrelease calculated by measuring the radioactivity in the supernatant(see page 176, column 1, line 33-column 2, line 17).

The Blaustein et al. method employs planar phospholipid bilayermembranes, which are used to test for channel forming activity. In moredetail, salt solutions on either side of the membrane are buffered at adifferent pH—on the cis side, pH 4.7 or 5.5 and on the trans side, pH7.4. The “conjugate” to be tested is added to the cis side of themembrane and electrical measurements are made under voltage clampconditions, in order to monitor the flow of current across the membrane(see paragraph 2.2, pages 116-118). The presence of an activetranslocation function is confirmed by a steady rate of channel turn-on(i.e. a positive result for channel formation) —see paragraph 3, page118.

Targeting Moiety (TM) function after conjugation may be tested byassaying for the agonist function inherent to the TM. Suitable methodsinclude those described in Example 1.

The ability of the conjugate of the invention to inhibit substance Prelease from nociceptive afferent cells can be assessed using themethods described in Example 15.

In Example 15, a nociceptin-LHN/A conjugate according to the firstaspect of the invention is assessed for its ability to inhibit therelease of substance P from primary nociceptive sensory afferentneurons. As can be seen from Table 1, incubation of the conjugate withcultures of nociceptive afferent neurons results in a significantinhibition of release of substance P (when compared to incubation of thecells with LHN/A alone). The experiment therefore confirms that theconjugate is inhibiting substance P release from these cells.

In use of the present invention, a pain-sensing target cell is selectedin which it is desired to reduce or inhibit the process of exocyticfusion, which exocytic process contributes to the symptoms associatedwith the sensation of pain. For example, the target cell in question maydemonstrate an undesirable phenotype (e.g. an undesirable secretion, orthe expression of an undesirable concentration of membrane receptor,transporter or membrane channel), which contributes to the symptomsassociated with pain. Alternatively, a target cell may be selected inwhich the process of exocytic fusion contributes to the sensation ofpain.

In preferred embodiments of the invention, the target cell is anociceptive sensory afferent cell, preferably a primary nociceptiveafferent cell (e.g. an A-fibre such as an Aδ-fibre or a C-fibre). Thus,the conjugates of the present invention are capable of inhibitingneurotransmitter or neuromodulator (e.g. glutamate, substance P,calcitonin-gene related peptide (CGRP), and/or neuropeptide Y) releasefrom discrete populations of nociceptive sensory afferent neurons. Inuse, the conjugates reduce or prevent the transmission of sensoryafferent signals (e.g. neurotransmitters or neuromodulators) fromperipheral to central pain fibres, and therefore have application astherapeutic molecules for the treatment of pain, in particular chronicpain.

It is routine to confirm that a TM binds to a nociceptive sensoryafferent. For example, a simple radioactive displacement experiment maybe employed in which tissue or cells representative of the nociceptivesensory afferent (for example DRGs) are exposed to labelled (e.g.tritiated) ligand in the presence of an excess of unlabelled ligand. Insuch an experiment, the relative proportions of non-specific andspecific binding may be assessed, thereby allowing confirmation that theligand binds to the nociceptive sensory afferent target cell.Optionally, the assay may include one or more binding antagonists, andthe assay may further comprise observing a loss of ligand binding.Examples of this type of experiment can be found in Hulme, E. C. (1990),Receptor-binding studies, a brief outline, pp 303-311, in Receptorbiochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford UniversityPress.

According to a second aspect, the present invention provides anon-cytotoxic conjugate for inhibition or reduction of exocytotic fusionin a nociceptive sensory afferent cell, comprising:

-   -   (i) a Targeting Moiety (TM),        -   wherein said TM is an agonist of a receptor that is present            on said nociceptive sensory afferent cell, and wherein said            receptor undergoes endocytosis to be incorporated into an            endosome within the nociceptive sensory afferent cell;    -   (ii) a DNA sequence encoding a non-cytotoxic protease or a        fragment thereof,        -   wherein the DNA sequence is expressible in the nociceptive            sensory afferent cell and when so expressed provides a            protease or protease fragment capable of cleaving a protein            of the exocytic fusion apparatus of said nociceptive sensory            afferent cell; and    -   (iii) a Translocation Domain,        -   wherein the Translocation Domain translocates the DNA            sequence encoding the protease or protease fragment from            within the endosome, across the endosomal membrane, and into            the nociceptive sensory afferent cell.

In a preferred embodiment, the receptor is an ORL₁ receptor.

DNA encoding a protein of interest can be transfected into eukaryoticcells through receptor-mediated endocytosis of a protein-DNA conjugate,as confirmed by Cotton et al. (Cotton, M., Wagner, E. and Birnstiel, L.(1993) Receptor-mediated transport of DNA into eukaryotic cells. Methodsin Enzymol. 217, 619-645). Several methods exist for condensing DNA to asuitable size using polycationic ligands. These include: polylysine,various cationic peptides and cationic liposomes. Of these, polylysinewas used in the present study because of its successfully reported usein receptor-mediated transfection studies (Cotton et al., 1993).

The DNA sequence encoding the non-cytotoxic protease component may beexpressed under the control of an operably linked promoter present aspart of the agent (e.g. as part of the protease DNA sequence upstream ofthe coding region). Alternatively, expression of the protease componentin the target cell may rely on a promoter present in the target cell.

The DNA sequence encoding the protease component may integrate into aDNA sequence of the target cell. One or more integration site(s) may beprovided as part of the conjugate (e.g. as part of the protease DNAsequence).

The TM, Translocation Domain and protease components of this secondaspect of the invention are as defined for the first aspect of theinvention. Examples 13 and 14 describe the preparation of conjugatesaccording to the second aspect of the invention.

According to a third aspect, the present invention provides apharmaceutical composition comprising a conjugate according to the firstand/or second aspect of the present invention.

The pharmaceutical composition may further comprise apharmaceutically-acceptable carrier, and/or a suitable diluent and/orexcipient, although the exact form of the composition may be tailored tothe mode of administration. Administration is preferably to a mammal,more preferably to a human.

The components of the composition may, for example, be employed in theform of an aerosol or nebulisable solution for inhalation or a sterilesolution for parenteral administration, intra-articular administrationor intra-cranial administration.

The composition may also be administered by i.v. injection, whichincludes the use of pump systems. Spinal injection (e.g. epidural orintrathecal) or indwelling pumps may also be used.

The dosage ranges for administration of the components of the presentinvention are those to produce the desired therapeutic effect. It willbe appreciated that the dosage range required depends on the precisenature of the components, the route of administration, the nature of theformulation, the age of the patient, the nature, extent or severity ofthe patient's condition, contraindications, if any, and the judgement ofthe attending physician.

Suitable daily dosages (for each component) are in the range 0.0001-1mg/kg, preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, andparticularly preferably 0.004-0.5 mg/kg. The unit dosage can vary fromless that 1 microgram to 30 mg, but typically will be in the region of0.01 to 1 mg per dose, which may be administered daily or preferablyless frequently, such as weekly or six monthly.

A particularly preferred dosing regimen is based on 2.5 ng of fusionprotein (e.g. CPNv/A) as the 1× dose. In this regard, preferred dosagesare in the range 1×-100× (i.e. 2.5-250 ng). This dosage range issignificantly lower (i.e. at least 10-fold, typically 100-fold lower)than would be employed with other types of analgesic molecules such asNSAIDS, morphine, and gabapentin. Moreover, the above-mentioneddifference is considerably magnified when the same comparison is made ona molar basis—this is because the fusion proteins of the presentinvention have a considerably greater Mw than do conventional ‘small’molecule therapeutics.

Wide variations in the required dosage, however, are to be expecteddepending on the precise nature of the components, and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection.

Variations in these dosage levels can be adjusted using standardempirical routines for optimisation, as is well understood in the art.

Compositions suitable for injection may be in the form of solutions,suspensions or emulsions, or dry powders which are dissolved orsuspended in a suitable vehicle prior to use.

Fluid unit dosage forms are typically prepared utilising a pyrogen-freesterile vehicle.

The active ingredients, depending on the vehicle and concentration used,can be either dissolved or suspended in the vehicle.

Solutions may be used for all forms of parenteral administration, andare particularly used for intravenous injection. In preparing solutionsthe components can be dissolved in the vehicle, the solution being madeisotonic if necessary by addition of sodium chloride and sterilised byfiltration through a sterile filter using aseptic techniques beforefilling into suitable sterile vials or ampoules and sealing.Alternatively, if solution stability is adequate, the solution in itssealed containers may be sterilised by autoclaving.

Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents and/orlocal anaesthetic agents may be dissolved in the vehicle.

Dry powders which are dissolved or suspended in a suitable vehicle priorto use may be prepared by filling pre-sterilised drug substance andother ingredients into a sterile container using aseptic technique in asterile area.

Alternatively the components of the composition may be dissolved in anaqueous vehicle, the solution is sterilized by filtration anddistributed into suitable containers using aseptic technique in asterile area. The product is then freeze-dried and the containers aresealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile components are suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The components may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone isincluded in the composition(s) to facilitate uniform distribution of thecomponents.

Compositions suitable for administration via the respiratory tractinclude aerosols, nebulisable solutions or microfine powders forinsufflation. In the latter case, particle size of less than 50 microns,especially less than 10 microns, is preferred. Such compositions may bemade up in a conventional manner and employed in conjunction withconventional administration devices.

The compositions described in this invention can be used in vivo, eitherdirectly or as a pharmaceutically acceptable salt, for the treatment ofconditions involving exocytosis (for example secretion, or the deliveryof proteins such as receptors, transporters, and membrane channels tothe plasma membrane of a cell).

According to a fourth aspect, the present invention provides a DNAconstruct that encodes a conjugate according to the first or secondaspects of the invention.

By expressing the construct in a host cell, conjugates of the inventionmay be prepared.

According to a fifth aspect, the present invention provides a method oftreatment of pain by administration to a patient of a conjugate,composition, or construct according to the first to fourth aspects ofthe invention, or any combination thereof.

In a preferred embodiment, the invention provides a method of treatingchronic pain.

According to a sixth aspect, the present invention provides for the useof a conjugate, composition or construct according to the first tofourth aspects of the invention, for the manufacture of a medicament fortreating pain, preferably chronic pain.

Definitions Section

Exocytic fusion is a process by which intracellular molecules aretransported from the cytosol of a pain-sensing target cell to the plasma(i.e. cell) membrane thereof. Thereafter, the intracellular moleculesmay become displayed on the outer surface of the plasma membrane, or maybe secreted into the extracellular environment.

In a healthy individual, the rate of exocytic fusion is carefullyregulated and allows control of the transport of molecules between thecytosol and the plasma membrane of a pain-sensing cell. For example,regulation of the exocytic cycle allows control of the density ofreceptors, transporters, or membrane channels present at the cell'ssurface, and/or allows control of the secretion rate of intracellularcomponents (e.g. neurotransmitters) from the cytosol of the cell.

However, in an unhealthy individual, the regulation of exocytic fusionmay be modified. For example, exocytic fusion may cause affectedpain-sensing cells to enter a state of hypersecretion. Alternatively,exocytic fusion may result in the display of an increased concentrationof receptors, transporters, or membrane channels present on the surfaceof the pain-sensing, which may expose the cell to undesirable externalstimuli. Thus, the process of exocytic fusion may contribute to theprogression and/or severity of pain, and therefore provides a target fortherapeutic intervention.

It should also be appreciated that otherwise normal rates of cellularexocytic fusion may contribute to the progression and severity of painin compromised patients. Thus, by targeting exocytic fusion inaccordance with the present invention, it is also possible to providetherapy in such patients

Targeting Moiety (TM) means any chemical structure associated with aconjugate that functionally interacts with a receptor, e.g. an ORL₁receptor, to cause a physical association between the conjugate and thesurface of a pain-sensing target cell. The term TM embraces any molecule(i.e. a naturally occurring molecule, or a chemically/physicallymodified variant thereof) that is capable of binding to a receptor onthe target cell, which receptor is capable of internalisation (e.g.endosome formation)—also referred to as receptor-mediated endocytosis.The TM may possess an endosomal membrane translocation domain, in whichcase separate TM and Translocation Domain components need not be presentin an agent of the present invention.

The term “fragment” means a peptide having at least thirty-five,preferably at least twenty-five, more preferably at least fifteen, andmost preferably at least ten amino acid residues of the TM in question.In one embodiment, the first amino acid residue of the fragment is theN-terminal amino acid residue of the TM from which the fragment has beenderived.

An example of a “variant” is a peptide or peptide fragment of a TM thatcontains one or more analogues of an amino acid (e.g. an unnatural aminoacid), or a substituted linkage.

A “derivative” comprises the TM in question, and a further peptidesequence. The further peptide sequence should preferably not interferewith the basic folding and thus conformational structure of the TM. Twoor more peptides (or fragments, or variants) may be joined together toform a derivative. Alternatively, a peptide (or fragment, or variant)may be joined to an unrelated molecule (e.g. a second, unrelatedpeptide). Derivatives may be chemically synthesized, but will betypically prepared by recombinant nucleic acid methods. Additionalcomponents such as lipid, and/or polysaccharide, and/or polyketidecomponents may be included.

The term non-cytotoxic means that the protease molecule in question doesnot kill the pain-sensing target cell to which it has been re-targeted.

The “protease cleavage site” of the present invention allows cleavage(preferably controlled cleavage) of the conjugate at a position betweenthe non-cytotoxic protease component and the TM component. In oneembodiment, the conjugate may include more than one proteolytic cleavagesite. However, where two or more such sites exist, they are different,thereby substantially preventing the occurrence of multiple cleavageevents in the presence of a single protease. In another embodiment, itis preferred that the conjugate has a single protease cleavage site. Theprotease cleavage sequence(s) may be introduced (and/or any inherentcleavage sequence removed) at the DNA level by conventional means, suchas by site-directed mutagenesis. Screening to confirm the presence ofcleavage sequences may be performed manually or with the assistance ofcomputer software (e.g. the MapDraw program by DNASTAR, Inc.).

Whilst any protease cleavage site may be employed, the following arepreferred:

Enterokinase (DDDDK↓) SEQ ID NO: 102 Factor Xa (IEGR↓/IDGR↓)SEQ ID NO: 103 TEV(Tobacco (ENLYFQ↓G) SEQ ID NO: 104 Etch virus)Thrombin (LVPR↓GS) SEQ ID NO: 105 PreScission (LEVLFQ↓GP).SEQ ID NO: 106

Also embraced by the term protease cleavage site is an intein, which isa self-cleaving sequence. The self-splicing reaction is controllable,for example by varying the concentration of reducing agent present.

The present invention is now described by reference to the followingExamples and Figures, without intended limitation thereto.

FIGURES

FIG. 1 Expression and purification of recLH_(N)/B fusion protein

FIG. 2 Expression and purification of LH_(N)/C fusion protein

FIG. 3 Expression and purification of N[1-17]-LH_(N)/A fusion protein

FIG. 4 Purification of a LC/A-nociceptin-H_(N)/A fusion protein

FIG. 5 Purification of a nociceptin-LC/A-H_(N)/A fusion protein

FIG. 6 Purification of a LC/C-nociceptin-H_(N)/C fusion protein

FIG. 7 Purification of a LC/A-met enkephalin-H_(N)/A fusion protein

FIG. 8 Comparison of binding efficacy of a LC/A-nociceptin-H_(N)/Afusion protein and a nociceptin-LC/A-H_(N)/A fusion protein

FIG. 9 In vitro catalytic activity of a LC/A-nociceptin-H_(N)/A fusionprotein

FIG. 10 Purification of a LC/A-nociceptin variant-H_(N)/A fusion protein

FIG. 11 Comparison of binding efficacy of a LC/A-nociceptin-H_(N)/Afusion protein and a LC/A-nociceptin variant-H_(N)/A fusion protein

FIG. 12 Expressed/purified LC/A-nociceptin-H_(N)/A fusion protein familywith variable spacer length product(s)

FIG. 13 Inhibition of SP release and cleavage of SNAP-25 by CPN-A

FIG. 14 Inhibition of SP release and cleavage of SNAP-25 over extendedtime periods after exposure of DRG to CPN-A

FIG. 15 Cleavage of SNAP-25 by CPNv-A

FIG. 16 Cleavage of SNAP-25 over extended time periods after exposure ofDRG to CPNv-A

FIG. 17 CPNv-A fusion-mediated displacement of [3H]-nociceptin binding

FIG. 18 Expressed/purified CPNv(Ek)-A product

FIG. 19 Cleavage of SNAP-25 by CPNv(Ek)-A

FIG. 20 Expressed/purified CPNv-C product

FIG. 21 Cleavage of syntaxin by CPNv-C

FIG. 22 CPN-A efficacy in the Acute Capsaicin-Induced MechanicalAllodynia model

FIG. 23 CPN-A efficacy in the Streptozotocin (STZ)-Induced PeripheralDiabetic Neuropathy (Neuropathic Pain) model

FIG. 24 CPNv-A efficacy in the Acute Capsaicin-Induced MechanicalAllodynia model

FIG. 25 Expressed/purified LC/A-CPLE-H_(N)/A product

FIG. 26 Expressed/purified LC/A-CPBE-H_(N)/A product

FIG. 27 Expressed/purified CPOP-A product

FIG. 28 Expressed/purified CPOPv-A product

FIG. 29 In vitro SNAP-25 cleavage in a DRG cell model

FIG. 30 Expressed/purified CPNv-A-FXa-HT (removable his-tag)

FIG. 31 In vitro efficacy of LC/A-nociceptin-H_(N)/A fusion proteinswith variable spacer length, as assessed by ligand competition assay

FIG. 32 In vitro efficacy of LC/A-nociceptin-H_(N)/A fusion proteinswith variable spacer length, as assessed by in vitro SNAP-25 cleavage

The Figures are now described in more detail.

FIG. 1—Expression and Purification of recLH_(N)/B Fusion Protein

SDS-PAGE analysis of expression and purification of recLH_(N)/B from E.coli. In FIG. 1, recLH_(N)/B is purified from cell paste using a threecolumn strategy as described in Example 3. Protein samples are separatedby SDS-PAGE and visualised by staining with simplyblue safestaincoomassie reagent. Crude, soluble MBP-LH_(N)/B fusion protein containedwithin the clarified extract (lane 2) is loaded onto Q-Sepharose FFanion-exchange resin. Lane 3 represents recombinant MBP-LH_(N)/B fusioneluted from column at 150-200 mM salt. This sample is treated withfactor Xa protease to remove MBP affinity tag (lane 4), and cleavedmixture diluted to lower salt concentration prior to loading onto aQ-Sepharose FF anion-exchange column. Material eluted between 120-170 mMsalt was rich in LH_(N)/B (lane 5). Protein in lanes 6 and 8 representsLH_(N)/B harvested after treatment with enterokinase and finalpurification using Benzamidine Sepharose, under non-reducing andreducing conditions respectively. Lanes 1 and 7 represent molecular massmarkers [Mark 12 (Invitrogen)].

FIG. 2—Expression and Purification of LH_(N)/C Fusion Protein

SDS-PAGE analysis of expression and purification of LH_(N)/C from E.coli. In FIG. 2, recLH_(N)/C is purified from E. coli cell paste using atwo-step strategy described in Example 4. Protein samples are separatedby SDS-PAGE and visualised by staining with coomassie blue. ClarifiedCrude cell lysate (lane 2) is loaded onto Q-Sepharose FF anion-exchangeresin. Fusion protein, MBP-LH_(N)/C is eluted with 0.1 M NaCl (lane 3).Eluted material incubated at 22° C. for 16 h with factor Xa protease(New England Biolabs) to cleave fusion tag MBP and nick recLH_(N)/C atthe linker site. The protein of interest is further purified fromcleaved fusion products (lane 4) using Q-Sepharose FF. Lanes 5 and 7show purified recLH_(N)/C under non-reducing conditions and reduced with10 mM DTT respectively, to illustrate disulphide bonding at the linkerregion between LC and H_(N) domains after nicking with factor Xa. Lanes1 and 6 represent molecular mass markers (shown in KDa); Mark 12(Invitrogen).

FIG. 3—Expression and Purification of N[1-17]-LH_(N)/A Fusion Protein

SDS-PAGE analysis of expression and purification of N[1-17]-LH_(N)/Afrom E. coli. In FIG. 3, N[1-17]-LH_(N)/A is purified from E. coli BL21cell paste using the methodology outlined in Example 9. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 4—Purification of a LC/A-nociceptin-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, a LC/A-nociceptin-H_(N)/Afusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 5—Purification of a Nociceptin-LC/A-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, a nociceptin-LC/A-H_(N)/Afusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 6—Purification of a LC/C-nociceptin-H_(N)/C Fusion Protein

Using the methodology outlined in Example 26, an LC/C-nociceptin-H_(N)/Cfusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 7—Purification of a LC/A-met Enkephalin-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, an LC/A-metenkephalin-H_(N)/A fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

FIG. 8—Comparison of Binding Efficacy of a LC/A-nociceptin-H_(N)/AFusion Protein and a Nociceptin-LC/A-H_(N)/A Fusion Protein

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin-H_(N)/A fusion is far superior to thenociceptin-LC/A-H_(N)/A fusion at interacting with the ORL₁ receptor.

FIG. 9—In vitro Catalytic Activity of a LC/A-nociceptin-H_(N)/A FusionProtein

The in vitro endopeptidase activity of the purifiedLC/A-nociceptin-H_(N)/A fusion protein was determined essentially asdescribed in Chaddock et al 2002, Prot. Express Purif. 25, 219-228.Briefly, SNAP-25 peptide immobilised to an ELISA plate was exposed tovarying concentrations of fusion protein for 1 hour at 37° C. Followinga series of washes, the amount of cleaved SNAP-25 peptide was quantifiedby reactivity with a specific antisera.

FIG. 10—Purification of a LC/A-nociceptin Variant-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, an LC/A-nociceptinvariant-H_(N)/A fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

FIG. 11—Comparison of Binding Efficacy of a LC/A-nociceptin-H_(N)/AFusion Protein and a LC/A-nociceptin Variant-H_(N)/A Fusion Protein

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin variant-H_(N)/A fusion (CPNv-LHA) is superior to theLC/A-nociceptin variant-H_(N)/A fusion (CPN-LHA) at interacting with theORL₁ receptor.

FIG. 12—Expressed/Purified LC/A-nociceptin-H_(N)/A Fusion Protein Familywith Variable Spacer Length Product(s)

Using the methodology outlined in Example 26, variants of theLC/A-CPN-H_(N)/A fusion consisting of GS10, GS30 and HX27 are purifiedfrom E. coli cell paste. Samples from the purification ofLC/A-CPN(GS10)-H_(N)/A, LC/A-CPN(GS15)-H_(N)/A, LC/A-CPN(GS25)-H_(N)/A,LC/A-CPN(GS30)-H_(N)/A and LC/A-CPN(HX27)-H_(N)/A were assessed bySDS-PAGE prior to staining with Coomassie Blue. The electrophoresisprofile indicates purification of a disulphide-bonded di-chain speciesof the expected molecular mass of CPBE-A. Top panel: M=benchmarkmolecular mass markers; S =total E. coli protein soluble fraction;FT=proteins that did not bind to the Ni²⁺-charged Sepharose column;FUSION=fusion protein eluted by the addition of imidazole. Bottom panel:Lane 1=benchmark molecular mass markers; Lane 2=total E. coli proteinsoluble fraction; Lane 3=purified material following initial capture onNi²⁺-charged Sepharose; Lane 4=Factor Xa treated material prior to finalcapture on Ni²⁺-charged Sepharose; Lane 5=purified final material postactivation with Factor Xa (5 μl); Lane 6=purified final material postactivation with Factor Xa (10 μl); Lane 7=purified final material postactivation with Factor Xa (20 μl); Lane 8=purified final material postactivation with Factor Xa+DTT (5 μl); Lane 9=purified final materialpost activation with Factor Xa+DTT (10 μl); Lane 10=purified finalmaterial post activation with Factor Xa+DTT (20 μl).

FIG. 13—Inhibition of SP Release and Cleavage of SNAP-25 by CPN-A

Briefly, primary cultures of dorsal root ganglia (DRG) were exposed tovarying concentrations of CPN-A for 24 hours. Cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis and plotted againstfusion concentration (dashed line). Material was also recovered for ananalysis of substance P content using a specific EIA kit. Inhibition ofsubstance P release is illustrated by the solid line. The fusionconcentration required to achieve 50% maximal SNAP-25 cleavage isestimated to be 6.30±2.48 nM.

FIG. 14—Inhibition of SP Release and Cleavage of SNAP-25 Over ExtendedTime Periods After Exposure of DRG to CPN-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT/A) wasused as a control. After this initial exposure, extracellular materialwas removed by washing, and the cells incubated at 37° C. for varyingperiods of time. At specific time points, cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis and illustrated by thedotted lines. Material was also recovered for an analysis of substance Pcontent using a specific EIA kit. Inhibition of substance P release isillustrated by the solid lines.

FIG. 15—Cleavage of SNAP-25 by CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-A for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitatean assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis. The fusion concentration requiredto achieve 50% maximal SNAP-25 cleavage is estimated to be 1.38±0.36 nM.

FIG. 16—Cleavage of SNAP-25 Over Extended Time Periods After Exposure ofDRG to CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-A for 24 hours. CPN-A was used as a control.After this initial exposure, extracellular material was removed bywashing, and the cells incubated at 37° C. for varying periods of time.At specific time points, cellular proteins were separated by SDS-PAGE,Western blotted, and probed with anti-SNAP-25 to facilitate anassessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis.

FIG. 17—CPNv-A Fusion-Mediated Displacement of [3H]-nociceptin Binding

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin variant-H_(N)/A fusion (labelled as CPNv-LHnA) issuperior to the LC/A-nociceptin-H_(N)/A fusion (labelled as CPN-LHnA) atinteracting with the ORL₁ receptor.

FIG. 18—Expressed/Purified CPNv(Ek)-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv(Ek)-A. Lane 1=benchmark molecular mass markers; Lane 2=total E.coli protein soluble fraction; Lane 3=purified material followinginitial capture on Ni²+-charged Sepharose; Lane 4=purified finalmaterial post activation with enterokinase (5 μl); Lane 5=purified finalmaterial post activation with enterokinase (10 μl); Lane 6=purifiedfinal material post activation with enterokinase (20 μl); Lane7=purified final material post activation with enterokinase+DTT (5 μl);Lane 8=purified final material post activation with enterokinase+DTT (10μl); Lane 9=purified final material post activation withenterokinase+DTT (20 μl).

FIG. 19—Cleavage of SNAP-25 by CPNv(Ek)-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv(Ek)-A for 24 hours. Cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis. CPNv-A as prepared inExample 26 was used for comparison purposes. The percentage cleavage ofSNAP-25 by CPNv(Ek)-A (labelled as En activated) and CPNv-A (labelled asXa activated) are illustrated.

FIG. 20—Expressed/Purified CPNv-C Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv-C. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purifiedmaterial following second capture on Ni²⁺-charged Sepharose; Lane6=final purified material; Lane 7=final purified material+DTT; Lane8=benchmark molecular mass markers.

FIG. 21—Cleavage of Syntaxin by CPNv-C

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-C for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-syntaxin tofacilitate an assessment of syntaxin cleavage. The percentage of cleavedsyntaxin was calculated by densitometric analysis. The fusionconcentration required to achieve 50% maximal syntaxin cleavage isestimated to be 3.13±1.96 nM.

FIG. 22—CPN-A Efficacy in the Acute Capsaicin-Induced MechanicalAllodynia Model

The ability of an LC/A-nociceptin-H_(N)/A fusion (CPN/A) to inhibitcapsaicin-induced mechanical allodynia was evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalswere evaluated for paw withdrawal frequency (PWF %) in response to a 10g Von Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat); after subcutaneous intraplantartreatment with CPN/A but before capsaicin (Pre-CAP); and followingcapsaicin challenge post-injection of CPN/A (average of responses at 15′and 30′; CAP).

Capsaicin challenge was achieved by injection of 10 μL of a 0.3%solution. Sample dilutions were prepared in 0.5% BSA/saline.

FIG. 23—CPN-A efficacy in the Streptozotocin (STZ)-Induced PeripheralDiabetic Neuropathy (Neuropathic Pain) Model

Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ incitrate buffer (I.V.) and blood glucose and lipid are measured weekly todefine the readiness of the model. Paw Withdrawal Threshold (PWT) ismeasured in response is to a Von Frey filament stimulus series over aperiod of time. Allodynia is said to be established when the PWT on twoconsecutive test dates (separated by 1 week) measures below 6 g on thescale. At this point, rats are randomized to either a saline group(negative efficacy control), gabapentin group (positive efficacycontrol) or a test group (CPN/A). Test materials (20-25 μ) are injectedsubcutaneously as a single injection (except gabapentin) and the PWT ismeasured at 1 day post-treatment and periodically thereafter over a 2week period. Gabapentin (30 mg/kg i.p. @3 ml/kg injection volume) isinjected daily, 2 hours prior to the start of PWT testing.

FIG. 24—CPNv-A Efficacy in the Acute Capsaicin-Induced MechanicalAllodynia Model

The ability of an LC/A-nociceptin variant-H_(N)/A fusion (CPNv/A) toinhibit capsaicin-induced mechanical allodynia was evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalswere evaluated for paw withdrawal frequency (PWF %) in response to a 10g Von Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat), after subcutaneous intraplantartreatment with CPNv/A but before capsaicin (Pre-CAP), and followingcapsaicin challenge post-injection of CPNv/A (average of responses at15′ and 30′; CAP). Capsaicin challenge was achieved by injection of 10μL of a 0.3% solution. Sample dilutions were prepared in 0.5%BSA/saline. These data are expressed as a normalized paw withdrawalfrequency differential, in which the difference between the peakresponse (post-capsaicin) and the baseline response (pre-capsaicin) isexpressed as a percentage. With this analysis, it can be seen thatCPNv/A is more potent than CPN/A since a lower dose of CPNv/A isrequired to achieve similar analgesic effect to that seen with CPN/A.

FIG. 25—Expressed/Purified LC/A-CPLE-H_(N)/A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species is of the expected molecular mass ofCPLE-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purifiedmaterial following second capture on Ni²⁺-charged Sepharose; Lane6=final purified material; Lane 7=final purified material+DTT.

FIG. 26—Expressed/Purified LC/A-CPBE-H_(N)/A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPBE-A. Lane 1=total E. coli protein soluble fraction; Lane 2=purifiedmaterial following initial capture on Ni²⁺-charged Sepharose; Lane3=Factor Xa treated material prior to final capture on Ni²+-chargedSepharose; Lane 4=purified final material post activation with Factor Xa(5 μl); Lane 5=purified final material post activation with Factor Xa(10 μl); Lane 6=purified final material post activation with Factor Xa(20 μ); Lane 7=purified final material post activation with FactorXa+DTT (5 μ); Lane 8=purified final material post activation with FactorXa+DTT (10 μ); Lane 9=purified final material post activation withFactor Xa+DTT (20 μ); Lane 10=benchmark molecular mass markers.

FIG. 27—Expressed/Purified CPOP-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPOP-A. Lane 1=benchmark molecular mass markers; Lane 2=purifiedmaterial following initial capture on Ni²⁺-charged Sepharose; Lane3=Factor Xa treated material prior to final capture on Ni²+-chargedSepharose; Lane 4=purified material following second capture onNi²+-charged Sepharose; Lane 5=purified final material post activationwith Factor Xa (5 μl); Lane 6=purified final material post activationwith Factor Xa (10 μl); Lane 7=is purified final material postactivation with Factor Xa (20 μl); Lane 8=purified final material postactivation with Factor Xa+DTT (5 μl); Lane 9=purified final materialpost activation with Factor Xa+DTT (10 μl); Lane 10=purified finalmaterial post activation with Factor Xa+DTT (20 μ).

FIG. 28—Expressed/Purified CPOPv-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPOPv-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purified finalmaterial post activation with Factor Xa (5 μl); Lane 6=purified finalmaterial post activation with Factor Xa (10 μl); Lane 7=purified finalmaterial post activation with Factor Xa (20 μ); Lane 8=purified finalmaterial post activation with Factor Xa+DTT (5 μl); Lane 9=purifiedfinal material post activation with Factor Xa+DTT (10 μ); Lane10=purified final material post activation with Factor Xa+DTT (20 μ).

FIG. 29—In vitro SNAP-25 Cleavage in a DRG Cell Model

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPOPv-A for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitatean assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis.

FIG. 30—Expressed/Purified CPNv-A-FXa-HT (Removable his-tag)

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species is of the expected molecular mass ofCPNv-A-FXa-HT. Lane 1=benchmark molecular mass markers; Lane 2=total E.coli protein soluble fraction; Lane 3=Factor Xa treated material priorto final capture on Ni²⁺-charged Sepharose; Lane 4=purified finalmaterial post activation with Factor Xa; Lane 5=purified final materialpost activation with Factor Xa+DTT.

FIG. 31—In vitro Efficacy of LC/A-nociceptin-H_(N)/A Fusion Proteinswith Variable Spacer Length, as Assessed by Ligand Competition Assay

The ability of LC/A-nociceptin-H_(N)/A fusions of variable spacer lengthto bind to the ORL₁ receptor was assessed using a simplecompetition-based assay. Primary cultures of dorsal root ganglia (DRG)were exposed to varying concentrations of test material in the presenceof 1 nM [3H]-nociceptin. The reduction in specific binding of theradiolabelled ligand was assessed by scintillation counting, and plottedin comparison to the efficacy of unlabelled ligand (Tocris nociceptin).The upper panel illustrates the displacement characteristics of the GS0,GS20, GS30 and Hx27 spacers, whilst the lower panel illustrates thedisplacement achieved by the GS10, GS15 and GS25 spaced fusion proteins.It is concluded that the GS0 and GS30 spacers are ineffective, and theGS10 is poorly effective, at displacing nociceptin from the ORL1receptor.

FIG. 32—In vitro Efficacy of LC/A-nociceptin-H_(N)/A Fusion Proteinswith Variable Spacer Length, as Assessed by in vitro SNAP-25 Cleavage

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPN-A (of variable spacer length) for 24 hours.Cellular proteins were separated by SDS-PAGE, Western blotted, andprobed with anti-SNAP-25 to facilitate an assessment of SNAP-25cleavage. The percentage of cleaved SNAP-25 was calculated bydensitometric analysis. The poorly effective binding characteristics ofthe GS10 spaced fusion protein (see FIG. 28) are reflected in the higherconcentrations of fusion required to achieve cleavage of intracellularSNAP-25. GS0 and GS30 spaced fusion proteins were completely ineffective(date not is shown). GS15, 20 and 25 spaced fusion proteins weresimilarly effective.

SEQ ID NOs:

-   SEQ ID NO:1 DNA sequence of N[1-17]-   SEQ ID NO:2 Protein Sequence of N[1-17]-   SEQ ID NO:3 DNA sequence of N[1-11]-   SEQ ID NO:4 Protein sequence of N[1-11]-   SEQ ID NO:5 DNA sequence of N[[Y10]1-11]-   SEQ ID NO:6 Protein sequence of N[[Y10]1-11]-   SEQ ID NO:7 DNA sequence of N[[Y11]1-11]-   SEQ ID NO:8 Protein sequence of N[[Y11]1-11]-   SEQ ID NO:9 DNA sequence of N[[Y14]1-17]-   SEQ ID NO:10 Protein sequence of N[[Y14]1-17]-   SEQ ID NO:11 DNA sequence of N[1-13]-   SEQ ID NO:12 Protein sequence of N[1-13]-   SEQ ID NO:13 DNA sequence of Nv (also known as N[[R14K15]1-17])-   SEQ ID NO:14 Protein sequence of Nv (also known as N[[R14K15]1-17])-   SEQ ID NO:15 DNA sequence of N[1-17]-LH_(N)/A fusion protein-   SEQ ID NO:16 Protein sequence of N[1-17]-LH_(N)/A fusion protein-   SEQ ID NO:17 DNA sequence of N[[Y11]1-11]-LHN/A fusion protein-   SEQ ID NO:18 Protein sequence of N[[Y11]1-11]-LHN/A fusion protein-   SEQ ID NO:19 DNA sequence of N[1-13]-LHN/A fusion protein-   SEQ ID NO:20 Protein sequence of N[1-13]-LHN/A fusion protein-   SEQ ID NO:21 DNA sequence of LHN/A-N[1-17] fusion protein-   SEQ ID NO:22 Protein sequence of LHN/A-N[1-17] fusion protein-   SEQ ID NO:23 DNA sequence of LHN/C-N[1-11] fusion protein-   SEQ ID NO:24 Protein sequence of LHN/C-N[1-11] fusion protein-   SEQ ID NO:25 DNA sequence of N[[Y14]1-17]-LHN/C fusion protein-   SEQ ID NO:26 Protein sequence of N[[Y14]1-17]-LHN/C fusion protein-   SEQ ID NO:27 DNA sequence of the LC/A-   SEQ ID NO:28 DNA sequence of the H_(N)/A-   SEQ ID NO:29 DNA sequence of the LC/B-   SEQ ID NO:30 DNA sequence of the H_(N)/B-   SEQ ID NO:31 DNA sequence of the LC/C-   SEQ ID NO:32 DNA sequence of the H_(N)/C-   SEQ ID NO:33 DNA sequence of the CPN-A linker-   SEQ ID NO:34 DNA sequence of the A linker-   SEQ ID NO:35 DNA sequence of the N-terminal presentation nociceptin    insert-   SEQ ID NO:36 DNA sequence of the CPN-C linker-   SEQ ID NO:37 DNA sequence of the CPBE-A linker-   SEQ ID NO:38 DNA sequence of the CPNvar-A linker-   SEQ ID NO:39 DNA sequence of the LC/A-CPN-H_(N)/A fusion-   SEQ ID NO:40 Protein sequence of the LC/A-CPN-H_(N)/A fusion-   SEQ ID NO:41 DNA sequence of the N-LC/A-H_(N)/A fusion-   SEQ ID NO:42 Protein sequence of the N-LC/A-H_(N)/A fusion-   SEQ ID NO:43 DNA sequence of the LC/C-CPN-H_(N)/C fusion-   SEQ ID NO:44 Protein sequence of the LC/C-CPN-H_(N)/C fusion-   SEQ ID NO:45 DNA sequence of the LC/C-CPN-H_(N)/C (A-linker) fusion-   SEQ ID NO:47 DNA sequence of the LC/A-CPME-H_(N)/A fusion-   SEQ ID NO:48 Protein sequence of the LC/A-CPME-H_(N)/A fusion-   SEQ ID NO:49 DNA sequence of the LC/A-CPBE-H_(N)/A fusion-   SEQ ID NO:50 Protein sequence of the LC/A-CPBE-H_(N)/A fusion-   SEQ ID NO:51 DNA sequence of the LC/A-CPNv-H_(N)/A fusion-   SEQ ID NO:52 Protein sequence of the LC/A-CPNv-H_(N)/A fusion-   SEQ ID NO:53 DNA sequence of the LC/A-CPN[1-11]-HN/A fusion-   SEQ ID NO:54 Protein sequence of the LC/A-CPN[1-11]-HN/A fusion-   SEQ ID NO:55 DNA sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusion-   SEQ ID NO:56 Protein sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusion-   SEQ ID NO:57 DNA sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusion-   SEQ ID NO:58 Protein sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusion-   SEQ ID NO:59 DNA sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion-   SEQ ID NO:60 Protein sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion-   SEQ ID NO:61 DNA sequence of the LC/A-CPN[1-13]-HN/A fusion-   SEQ ID NO:62 Protein sequence of the LC/A-CPN[1-13]-HN/A fusion-   SEQ ID NO:63 DNA sequence of the nociceptin-spacer-LC/A-H_(N)/A    fusion-   SEQ ID NO:64 Protein sequence of the nociceptin-spacer-LC/A-H_(N)/A    fusion-   SEQ ID NO:65 DNA sequence of the CPN-A GS10 linker-   SEQ ID NO:66 DNA sequence of the CPN-A GS15 linker-   SEQ ID NO:67 DNA sequence of the CPN-A GS25 linker-   SEQ ID NO:68 DNA sequence of the CPN-A GS30 linker-   SEQ ID NO:69 DNA sequence of the CPN-A HX27 linker-   SEQ ID NO:70 DNA sequence of the LC/A-CPN(GS15)-H_(N)/A fusion-   SEQ ID NO:71 Protein sequence of the LC/A-CPN(GS15)-H_(N)/A fusion-   SEQ ID NO:72 DNA sequence of the LC/A-CPN(GS25)-H_(N)/A fusion-   SEQ ID NO:73 Protein sequence of the LC/A-CPN(GS25)-H_(N)/A fusion-   SEQ ID NO:74 DNA sequence of the CPNvar-A Enterokinase activatable    linker-   SEQ ID NO:75 DNA sequence of the LC/A-CPNv(Ek)-H_(N)/A fusion-   SEQ ID NO:76 Protein sequence of the LC/A-CPNv(Ek)-H_(N)/A fusion-   SEQ ID NO:77 DNA sequence of the CPNvar-A linker-   SEQ ID NO:79 Protein sequence of the LC/C-CPNv-H_(N)/C fusion (act.    A)-   SEQ ID NO:80 DNA sequence of the LC/A-CPLE-H_(N)/A fusion-   SEQ ID NO:81 Protein sequence of the LC/A-CPLE-H_(N)/A fusion-   SEQ ID NO:82 DNA sequence of the LC/A-CPOP-H_(N)/A fusion-   SEQ ID NO:83 Protein sequence of the LC/A-CPOP-H_(N)/A fusion-   SEQ ID NO:84 DNA sequence of the LC/A-CPOPv-H_(N)/A fusion-   SEQ ID NO:85 Protein sequence of the LC/A-CPOPv-H_(N)/A fusion-   SEQ ID NO:86 DNA sequence of the IgA protease-   SEQ ID NO:87 DNA sequence of the IgA-CPNv-H_(N)/A fusion-   SEQ ID NO:88 Protein sequence of the IgA-CPNv-H_(N)/A fusion-   SEQ ID NO:89 DNA sequence of the FXa-HT-   SEQ ID NO:90 DNA sequence of the CPNv-A-FXa-HT-   SEQ ID NO:91 Protein sequence of the CPNv-A-FXa-HT fusion-   SEQ ID NO:92 DNA sequence of the DT translocation domain-   SEQ ID NO:93 DNA sequence of the CPLE-DT-A-   SEQ ID NO:94 Protein sequence of the CPLE-DT-A fusion-   SEQ ID NO:95 DNA sequence of the TeNT LC-   SEQ ID NO:96 DNA sequence of the CPNv-TENT LC-   SEQ ID NO:97 Protein sequence of the CPNV-TeNT LC fusion-   SEQ ID NO:98 DNA sequence of the CPNvar-C linker-   SEQ ID NO:99 DNA sequence of the LC/C-CPNv-H_(N)/C fusion (act. C)-   SEQ ID NO:100 Protein sequence of the LC/C-CPNv-H_(N)/C fusion (act.    C)

EXAMPLES Example 1 Confirmation of TM Agonist Activity by MeasuringRelease of Substance P from Neuronal Cell Cultures

Materials

Substance P EIA is obtained from R&D Systems, UK.

Methods

Primary neuronal cultures of eDRG are established as describedpreviously (Duggan et al., 2002). Substance P release from the culturesis assessed by EIA, essentially as described previously (Duggan et al.,2002). The TM of interest is added to the s neuronal cultures(established for at least 2 weeks prior to treatment); control culturesare performed in parallel by addition of vehicle in place of TM.Stimulated (100 mM KCl) and basal release, together with total celllysate content, of substance P are obtained for both control and TMtreated cultures. Substance P immunoreactivity is measured usingSubstance P Enzyme Immunoassay Kits (Cayman Chemical Company, USA or R&DSystems, UK) according to manufacturers' instructions.

The amount of Substance P released by the neuronal cells in the presenceof the TM of interest is compared to the release obtained in thepresence and absence of is 100 mM KCl. Stimulation of Substance Prelease by the TM of interest above the basal release, establishes thatthe TM of interest is an “agonist ligand” as defined in thisspecification. If desired the stimulation of Substance P release by theTM of interest can be compared to a standard Substance P release-curveproduced using the natural ORL-1 receptor ligand, nociceptin (Tocris).

Example 2 Expression and Purification of Catalytically Active LH_(N)/A

Materials

Synthetic DNA obtained from Sigma Genosys.

Restriction enzymes obtained from New England Biolabs.

Methods

The expression and purification of catalytically active LH_(N)/A wascarried out essentially as described in Sutton et al., (2005), Prot.Express. Purif., 40, pp 31-41.

Briefly, DNA encoding the light chain plus 423 amino acids from theN-terminal of the heavy chain of BoNT/A was synthesised by Sigma-Genosysto produce a synthetic LH_(N)/A gene with an E. coli codon bias. Thelinker region between the light chain and H_(N) domain was engineered tocontain a Factor Xa cleavage site by splice-overlap extension PCR. TwoPCR products were generated using primer pairs consisting of a long,mutagenic primer and a shorter, non-mutagenic primer:

(5′-tccaaaactaaatctctgATAGAAGGTAGAaacaaagcgctgaacgac; SEQ ID NO:107)with (5′-CTTGATGTACTCTGTGAACGTGCTC; SEQ ID NO:108); and

(5′-gtcgttcagcgctttgttTCTACCTTCTATcagagatttagttttgga; SEQ ID NO:109)with (5′-ATGGAGTTCGTTAACAAACAGTTC; SEQ ID NO:110).

The products from these two reactions were used as templates for thesplice-overlap extension PCR. A further PCR reaction was set up to addBamHI and SalI sites at either end of the activatable recLH_(N)/A geneand these sites were used for insertion is into an Invitrogen gatewayentry vector. The entry vector was then used, along with a gatewayrecombination site adapted pMAL c2x, in a LR clonase reaction to formpMAL c2x recLH_(N)/A. The pMAL c2x recLH_(N)/A was modified toincorporate a 6′HIS tag at the N-terminus of the MBP. This was achievedby the insertion of annealed oligonucleotides encoding the HIS tag intothe NdeI site of pMAL.

The expression vector expressing LH_(N)/A was transformed into E. coliHMS174 or AD494(DE3) (Novagen). Cultures were grown in Terrific brothcomplex medium supplemented with ZnCl₂ (1 μM), ampicillin (100 μg/ml),0.2% (w/v) glucose. Parameters for expression of all the constructs wereinitially determined in shake flask cultures before transferring into 8L fermentor systems. Starter cultures were grown for 16 hours at 37° C.,220 rpm and used to inoculate 1 L in which growth was continued at 37°C., 250 rpm. At an OD600 nm of 0.6 the temperature was reduced to 25° C.for 30 minutes before induction with 1 mM IPTG. Induction was continuedfor 4 hours before the cells were harvested and stored at −70° C.

Typically 16 g of cell paste was suspended in 160 ml PBS and lysed bysonication (MSE Soniprep 150). The resulting lysate was clarified bycentrifugation prior loading onto a 25 ml amylose column and eluted with10 mM maltose in PBS. The eluant contained approx. 50% pure fusionprotein and was treated with Factor Xa (1 unit Factor Xa/100 μg fusionprotein; 20 hours; 26° C.) to remove the HISMBP and cleave the LC-H_(N)junction to activate the protein. After incubation the sample was sfiltered (0.45 mm) and diluted two fold with water to give a 0.5× PBSbuffer composition. The cleaved, filtered and diluted recLH_(N)/A wasprocessed through a Q Sepharose FF column (10 ml) and eluted with a stepgradient of 80 mM NaCl containing HISMBP and 120 mM NaCl containingapprox. 75% pure recLH_(N)/A. The addition of His tag to MBP overcameprevious co-elution problems with LH_(N)/A and MBP. As a final polishingstep to ensure complete removal of the HISMBP, the 120 mM NaCl elutionfrom the Q Sepharose column was passed through a Nickel charged 5 mlHisTrap column (Amersham). The flow through from the HisTrap columncontained approx. 95% pure recLH_(N)/A (see the Figures in Sutton etal., (2005), Prot. Express. Purif., 40, pp 31-41 for an illustration ofthe purification is scheme for LHN/A).

Example 3 Expression and Purification of Catalytically ActiveRecombinant LH_(N)/B

The methodology described below will purify catalytically activeLH_(N)/B protease from

E. co/itransformed with the appropriate plasmid encoding the LH_(N)/Bpolypeptide. It should be noted that various sequences of suitableLH_(N)/B polypeptides have been described in PCT/GB97/02273, grantedU.S. Pat. No. 6,461,617 and U.S. patent application Ser. No. 10/241,596,incorporated herein by reference.

Methods

The coding region for LH_(N)/B is inserted in-frame to the 3′ of thegene encoding maltose binding protein (MBP) in the expression vectorpMAL (New England Biolabs) to create pMAL-c2x-LH_(N)/B. In thisconstruct, the expressed MBP and LH_(N)/B polypeptides are separated bya Factor Xa cleavage site, and the LC and H_(N) domains are separated bya peptide that is susceptible to cleavage with enterokinase. Theexpression clone is termed pMAL-c2X-synLH_(N)/B.

pMAL-c2X-synLH_(N)/B is transformed into E. coli HMS174 and cultured inTerrific broth complex medium in 8 L fermentor systems. Pre-inductionbacterial growth is maintained at 37° C. to an OD600 nm of 5.0, at whichstage expression of recMBP-LH_(N)/B is induced by addition of IPTG to0.5 mM and a reduction in temperature to 30° C. After four hours at 30°C. the bacteria are harvested by centrifugation and the resulting pastestored at −70° C.

The cell paste is resuspended in 20 mM Hepes pH 7.2, 125 mM NaCl, 1 μMZnCl₂ and cell disruption achieved using an APV-Gaulin lab model 1000homogeniser or a MSE Soniprep 150 sonicator. The resulting suspension isclarified by centrifugation prior to purification.

Following cell disruption, the MBP-fusion protein is captured either onan amylose affinity resin in 20 mM Hepes pH 7.2, 125 mM NaCl, 1 μMZnCl₂, or on a Q-Sepharose FF anion-exchange resin in 50 mM Hepes pH7.2, 1 μM ZnCl₂ with no salt. A single peak is eluted from the amyloseresin in the same buffer plus 10 mM maltose and from the Q-Sepharose in150-200 mM salt. Cleavage of the MBP-LH_(N)/B junction is completed inan 18 hours incubation step at 22° C. with Factor Xa (NEB) at 1 U/50 μgfusion protein. A substrate (MBP-LH_(N)/B) concentration of at least 4mg/ml is desirable for efficient cleavage to take place.

The cleaved protein is diluted with 20 mM Hepes to a buffer compositionof 20 mM Hepes, 25 mM NaCl, 1 μM ZnCl₂, pH 7.2 and processed through a QSepharose column to separate the MBP from LH_(N)/B. The LH_(N)/B iseluted from the Q-Sepharose column with 120-170 mM salt. The linkerbetween the light chain and H_(N) domain is then nicked by incubationwith enterokinase at 1 U/100 μg of LH_(N)/B at 22° C. for 16 hours.Finally, the enterokinase is separated from the nicked LH_(N)/B andother contaminating proteins on a Benzamidine Sepharose column, theenzyme preferentially binding to the resin over an incubation of 30minutes at 4° C. Purified LH_(N)/B is stored at −20° C. until required.See FIG. 1 for an illustration of the purification scheme forrecLH_(N)/B.

Example 4 Expression and Purification of Catalytically ActiveRecombinant LH_(N)/C

The coding region for LH_(N)/C is inserted in-frame to the 3′ of thegene encoding maltose binding protein (MBP) in the expression vectorpMAL (New England Biolabs) to create pMAL-c2x-LH_(N)/C. In thisconstruct the expressed MBP and LH_(N)/C polypeptides are separated by aFactor Xa cleavage site.

pMAL-c2x-LH_(N)/C is transformed into E. coli AD494 (DE3, IRL) andcultured in Terrific broth complex medium in 8 L fermentor systems.Pre-induction bacterial growth are maintained at 30° C. to an OD600 nmof 8.0, at which stage expression of recMBP-c2x-LH_(N)/C is induced byaddition of IPTG to 0.5 mM and a reduction in temperature of culture to25° C. After 4 hours at 25° C. the bacteria are harvested bycentrifugation and the resulting paste stored at −70° C.

The cell paste is resuspended in 50 mM Hepes pH 7.2, 1 μM ZnCl₂ at 1:6(w/v) and cell disruption is achieved using an APV-Gaulin lab model 1000homogeniser or a MSE Soniprep 150 sonicator. The resulting suspension isclarified by centrifugation prior to purification.

Following cell disruption and clarification, the MBP-fusion protein isseparated on a Q-Sepharose Fast Flow anion-exchange resin in 50 mM HepespH 7.2, 1 μM ZnCl₂ and eluted with the same buffer plus 100 mM NaCl. Adouble point cleavage is performed at the MBP-LH_(N)/C junction and theH_(N)-LC linker in a single incubation step with Factor Xa. The reactionis completed in a 16-hour incubation step at 22° C. with Factor Xa (NEB)at 1 U/100 ig fusion protein. The cleaved protein is diluted with 20 mMHepes to a buffer composition of 20 mM Hepes, 25 mM NaCl, pH 7.2 andprocessed through a second Q-Sepharose column to separate the MBP fromLH_(N)/C. Activated (disulphide-bonded cleaved linker) LH_(N)/C iseluted from the Q-Sepharose column by a salt gradient (20 mM Hepes, 500mM NaCl, 1 μM ZnCl₂, pH 7.2) in 120-170 mM salt. See FIG. 2 for anillustration of the purification of LH_(N)/C.

Example 5 Production of a Chemical Conjugate of Nociceptin and LH_(N)/A

Materials

C-terminally extended nociceptin peptide obtained from Sigma Genosys.Conjugation chemicals obtained from Pierce.

Methods

In order to couple the nociceptin peptide via a C-terminal Cys, thepeptide was first synthesised (by standard procedures, commerciallyobtainable) to include a Cys as the final C-terminal amino acid.

This peptide was then used as the second component in a sulphydryl basedis coupling reaction as described below (see also previous publicationsWO 99/17806 and WO 96/33273 and Duggan et al., (2002), J. Biol. Chem.277, 24846-34852 and Chaddock et al., (2000), Infect Immun., 68,2587-2593).

Sulphydryl Based Coupling Reaction

Briefly, approximately two reactive leaving groups were introduced intoLH_(N)/A (5 mg/ml in phosphate-buffered saline) by reaction withN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).

Derivatised material was isolated from excess SPDP by size exclusionchromatography. Reconstituted cysteine-tagged nociceptin ligand wasmixed with the derivatised LH_(N)/A in a 4:1 molar ratio, and incubatedat room temperature for 1 hour with gentle agitation in order to createa chemical conjugate through a reducible covalent disulphide bond.Initial fractionation of the conjugate mixture to remove unconjugatedpeptide was performed by size exclusion chromatography (Superose-12, orSuperdex G-200 depending on scale of conjugation).

Example 6 Production of a Chemical Conjugate of Nociceptin and LH_(N)/B

Materials

C-terminally extended nociceptin peptide obtained from Sigma Genosys.Conjugation chemicals obtained from Pierce.

Methods

Lyophilised nociceptin was dissolved by the addition of water anddialysed into MES buffer (0.1 M MES, 0.1 M NaCl, pH 5.0). To thissolution (at a concentration of about 0.3 mg/ml) was added PDPH (100mg/ml in DMF) to a final concentration of 1 mg/ml. After mixing, solidEDAC was added to produce a final concentration of about 0.2 mg/ml. Thereaction was allowed to proceed for at least 30 minutes at roomtemperature. Excess PDPH was then removed by desalting over a PD-10 iscolumn (Pharmacia) previously equilibrated with MES buffer.

An amount of LH_(N)/B equivalent to half the weight of nociceptin useddissolved in triethanolamine buffer (0.02 M triethanolamine/HCl, 0.1 Msodium chloride, pH 7.8) at a concentration of about 1 mg/ml, wasreacted with Traut's reagent (100 mM stock solution in 1 Mtriethanolamine/HCl, pH 8.0) at a final concentration of 2 mM. After 1hour, the LH_(N)/B was desalted into PBSE (phosphate buffered salinewith 1 mM EDTA) using a PD-10 column (Pharmacia). The protein peak fromthe column eluate was concentrated using a Microcon 50 (Amicon) to aconcentration of about 2 mg/ml.

The derivatised nociceptin was subjected to a final concentration stepresulting in a reduction in volume to less than 10% of the startingvolume and then mixed with the derivatised LH_(N)/B overnight at roomtemperature. The products of the reaction were analysed bypolyacrylamide gel electrophoresis in the presence of sodiumdodecyl-sulphate (SDS-PAGE).

The conjugate resulting from the above reaction was partially purifiedby size exclusion chromatography over Bio-Gel P-100 (BioRad). Theelution profile was followed by measuring the optical density at 280 nmand SDS-PAGE analysis of the fractions. This allowed the separation ofconjugate from free nociceptin and by-products of the reaction.

Example 7 Production of a Chemical Conjugate of Nociceptin 1-11 andLH_(N)/B

Materials

C-terminally extended nociceptin 1-11 peptide obtained from SigmaGenosys. Conjugation chemicals obtained from Pierce.

Methods

In order to couple the nociceptin 1-11 peptide via a C-terminal Cys, thepeptide was first synthesised (by standard procedures, commerciallyobtainable) to include a is Cys as the final C-terminal amino acid.

This peptide was then used as the second component in a sulphydryl basedcoupling reaction as described in Example 5.

Example 8 Production of a Chemical Conjugate of Nociceptin N[[Y14]1-17]and LH_(N)/C

Materials

C-terminally extended nociceptin N[[Y14]1-17] peptide obtained fromSigma Genosys.

Conjugation chemicals obtained from Pierce.

Methods

In order to couple the peptide via a C-terminal Cys, the peptide wasfirst synthesised (by standard procedures, commercially obtainable) toinclude a Cys as the final C-terminal amino acid.

This peptide was then used as the second component in a sulphydryl basedcoupling reaction as described in Example 5.

Example 9 Recombinant Production of a Single Polypeptide Fusion ofNociceptin-LH_(N)/A (SEQ ID NO:15 and SEQ ID NO:16)

The DNA sequence for the nociceptin-LH_(N)/A was designed by backtranslation of the LC/A, H_(N)/A, and nociceptin amino acid sequences.The complete ORF containing the nociceptin-LC/A-activation loop-H_(N)/Asequence was assembled within standard DNA sequence manipulationsoftware (EditSeq). The activation loop between the LC/A cysteine andthe H_(N)/A cysteine (CVRGIITSKTKSLDKGYNKALNDLC; SEQ ID NO:111) wasmodified to incorporate a Factor Xa protease recognition site.

Restriction sites appropriate to facilitate cloning into the requiredexpression vector (for example BamHI/SalI) were incorporated at the 5′and 3′ ends respectively of the sequence maintaining the correct readingframe. The DNA sequence was screened (using software such as MapDraw,DNASTAR Inc.) for restriction enzyme cleavage sequences incorporatedduring the back translation. Any cleavage sequences that were found tobe common to those required by the cloning system were removed manuallyfrom the proposed coding sequence ensuring common E. coli codon usagewas maintained. E. coli codon usage was assessed by reference tosoftware programs such as Graphical Codon Usage Analyser (Geneart), andthe % GC content and codon usage ratio assessed by reference topublished codon usage tables (for example GenBank Release 143, 13 Sep.2004).

This optimised DNA sequence containing the nociceptin-LC/A-activationloop-H_(N)/A open reading frame (ORF) was then commercially synthesizedand provided in the pCR 4 vector.

The DNA encoding the nociceptin-LH_(N)/A fusion was isolated from pCR 4and transferred into pMAL vector backbone to facilitate proteinexpression. The resultant pMAL NO-LHN/A vector was transformed intocompetent E. coli BL21 and correct transformants selected. A singlecolony of pMAL NO-LH_(N)/A was grown in Terrific broth complex mediumsupplemented with ZnCl₂ (1 mM), ampicillin (100 μg/ml), 0.2% (w/v)glucose. Expression of the insert was induced by the addition of IPTG(0.1 mM) and the culture maintained at 16° C. for 16 hours. After thisperiod of expression the bacteria were isolated by centrifugation andthe cell pellet stored at −20° C. until use.

10 g of E. coli BL21 cell paste was defrosted in a falcon tubecontaining 25 ml 50 mM HEPES, pH 7.2, 200 mM NaCl. The thawed cell pastewas made up to 80 ml with 50 mM HEPES, pH 7.2, 200 mM NaCl and sonicatedon ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22microns ensuring the sample remained cool. The lysed cells werecentrifuged at 18 000 rpm, 4° C. for 30 minutes. The supernatant wasloaded onto a 0.1 M NiSO₄ charged chelating column (20-30 ml column issufficient) and equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl.

Using a step gradient of 10 and 40 mM imidazol, the non-specific boundprotein was washed away and the fusion protein eluted with 100 mMimidazol. The eluted fusion protein was dialysed against 5 L of 50 mMHEPES, pH 7.2, 200 mM NaCl at 4° C. overnight and the OD of the dialysedfusion protein measured. 1 unit of Factor Xa was added per 100 μg fusionprotein and incubated at 25° C. static overnight. The cleavage mixturewas loaded onto a 0.1 M NiSO₄ charged Chelating column (20-30 ml columnis sufficient) and equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl.

Using a step gradient of 10 and 40 mM imidazol, the non-specific boundprotein was washed away and the fusion protein eluted with 100 mMimidazol. The eluted fusion protein was dialysed against 5 L of 50 mMHEPES, pH 7.2, 200 mM NaCl at 4° C. overnight and the fusionconcentrated to about 2 mg/ml, aliquoted and stored at −20° C.

FIG. 3 shows the SDS-PAGE analysis of expression and purification ofN[1-17]-LH_(N)/A .

Example 10 Recombinant Production of a Single Polypeptide Fusion of(Nociceptin 1-11)-LH_(N)/B

The DNA sequence for the (nociceptin 1-11)-LH_(N)/B was designed by backtranslation of the LC/B, H_(N)/B, and nociceptin 1-11 amino acidsequences. The complete ORF containing the(nociceptin1-11)-LC/B-activation loop-H_(N)/B sequence was assembledwithin standard DNA sequence manipulation software (EditSeq). Theactivation loop between the LC/B cysteine and the H_(N)/B cysteine wasmodified to incorporate a Factor Xa protease recognition site.

The recombinant fusion protein was then produced essentially asdescribed in Example 9.

Example 11 Recombinant Production of a Single Polypeptide Fusion of(Nociceptin N[[Y14]1-17])—LH_(N)/C (SEQ ID NO:25 and SEQ ID NO:26)

The DNA sequence for the nociceptin N[[Y14]1-17] was designed by backtranslation of the LC/C, H_(N)/C, and nociceptin N[[Y14]1-17] amino acidsequences. The complete ORF containing the (nociceptinN[[Y14]1-17])-LC/C-activation loop-H_(N)/C sequence was assembled withinstandard DNA sequence manipulation software (EditSeq). The activationloop between the LC/C cysteine and the H_(N)/C cysteine was modified toincorporate a Factor Xa protease recognition site.

The recombinant fusion protein was then produced essentially asdescribed in Example 9.

Example 12 Recombinant Production of a Single Polypeptide Fusion ofLH_(N)/C-(Nociceptin 1-11) (SEQ ID NO:23 and SEQ ID NO:24)

The DNA sequence for the LH_(N)/C-(nociceptin 1-11) was designed by backtranslation of the LC/C, H_(N)/C and nociceptin 1-11 amino acidsequences. The complete ORF (SEQ ID NO:23) containing theLC/C-activation loop-H_(N)/C-flexible spacer-(nociceptin 1-11) wasassembled within standard DNA sequence manipulation software (EditSeq).

The recombinant fusion protein (SEQ ID NO:24) was then producedessentially as described in Example 9.

Example 13 Production of a Conjugate for Delivery of DNA Encoding LC/CInto a Cell

The construction of a nociceptin-H_(N)-[LC/C] conjugate is describedbelow, where [LC/C] represents the polylysine condensed DNA encoding thelight chain of botulinum neurotoxin type C.

Materials

SPDP is from Pierce Chemical Co.

Additional reagents are obtained from Sigma Ltd.

Methods

Using a plasmid containing the gene encoding LC/C under the control of aCMV (immediate early) promoter, condensation of DNA was achieved usingSPDP-derivatised polylysine to a ratio of 2 DNA to 1 polylysine.Conjugates were then prepared by mixing condensed DNA (0.4 mg/ml) withH_(N)-nociceptin (100 μg/ml) for 16 h at 25° C. The SPDP-derivatisedpolylysine and the free —SH group present on the H_(N) domain combine tofacilitate covalent attachment of the DNA and protein.

Example 14 Production of a Conjugate for Delivery of DNA Encoding LC/BInto a Cell

The construction of a (nociceptin 1-11)-H_(N)-[LC/B] conjugate isdescribed below, where [LC/B] represents the polylysine condensed DNAencoding the light chain of botulinum neurotoxin type B.

Materials

SPDP is from Pierce Chemical Co. Additional reagents are obtained fromSigma Ltd.

Methods

Using a plasmid containing the gene encoding LC/B under the control of aCMV (immediate early) promoter, condensation of DNA was achieved usingSPDP-derivatised polylysine to a ratio of 2 DNA to 1 polylysine.Conjugates were then prepared by mixing condensed DNA (0.4 mg/ml) withH_(N)-(nociceptin 1-11) (100 μg/ml) for 16 h at 25° C. TheSPDP-derivatised polylysine and the free —SH group present on the H_(N)domain combine to facilitate covalent attachment of the DNA and protein.

Example 15 Assessment of the Activity of Nociceptin-LH_(N)/A inSubstance P Releasing Neuronal Cells

Using methodology described in Duggan et al., (2002, J. Biol. Chem.,277, 34846-34852), the activity of nociceptin-LH_(N)/A in substance Preleasing neuronal cells was assessed.

Nociceptin-LH_(N)/A fusion protein was applied to 2-week old dorsal rootganglia neuronal cultures, and incubated at 37° C. for 16 hours.Following the incubation, the media was removed and the ability of thecells to undergo stimulated release of substance P (SP) was assessed.

The release of SP from the neuronal cells incubated with thenociceptin-LH_(N)/A fusion protein was assayed in comparison to (i)LH_(N)/A-only treated cells and (ii) cells treated with media alone.This allowed the % inhibition of substance P from the eDRG to becalculated. The ability of the nociceptin-LH_(N)/A fusion protein toinhibit SP release (relative to cells treated with media alone) wasreported in Table 1. The data represent the mean of 3 determinations:

TABLE 1 nociceptin-LH_(N)/A fusion protein LH_(N)/A-only Test Material(μM) % Inhibition % Inhibition 1.0 47.3 25.6 0.1 13.8 −11.5

Example 16 Confirmation of ORL₁ Receptor Activation by MeasuringForskolin-Stimulated cAMP Production

Confirmation that a given TM is acting via the ORL₁ receptor is providedby the following test, in which the TMs ability to inhibitforskolin-stimulated cAMP production is assessed.

Materials

[³H]adenine and [¹⁴C]cAMP are obtained from GE Healthcare

Methods

The test is conducted essentially as described previously by Meunier etal. [Isolation and structure of the endogenous agonist of opioidreceptor-like ORL₁ receptor. Nature 377: 532-535, 1995] in intacttransfected-CHO cells plated on 24-well plastic plates.

To the cells is added [3H]adenine (1.0 μCi) in 0.4 ml of culture medium.The cells remain at 37° C. for 2 h to allow the adenine to incorporateinto the intracellular ATP pool. After 2 h, the cells are washed oncewith incubation buffer containing: 130 mM NaCl, 4.8 mM KCl, 1.2 mMKH₂PO₄, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 10 mM glucose, 1 mg/ml bovine serumalbumin and 25 mM HEPES, pH 7.4, and replaced with buffer containingforskolin (10 μM) and isobutylmethylxanthine (50 μM) with or without theTM of interest. After 10 min., the medium is aspirated and replaced with0.5 ml, 0.2 M HCl. Approximately 1000 cpm of [¹⁴C]cAMP is added to eachwell and used as an internal standard. The contents of the wells arethen transferred to columns of 0.65 g dry alumina powder. The columnsare eluted with 4 ml of 5 mM HCl, 0.5 ml of 0.1 M ammonium acetate, thentwo additional millilitres of ammonium acetate. The final eluate iscollected into scintillation vials and counted for ¹⁴C and tritium.Amounts collected are corrected for recovery of [¹⁴C]cAMP. TMs that areagonists at the ORL₁ receptor cause a reduction in the level of cAMPproduced in response to forskolin.

Example 17 Confirmation of ORL₁ Receptor Activation Using a GTPγSBinding Functional Assay

Confirmation that a given TM is acting via the ORL₁ receptor is alsoprovided by the is following test, a GTPγS binding functional assay.

Materials

[³⁵S]GTPγS is obtained from GE Healthcare

Wheatgerm agglutinin-coated (SPA) beads are obtained from GE Healthcare

Methods

This assay is carried out essentially as described by Traynor andNahorski [Modulation by μ-opioid agonists of guanosine-5-O-(3-[³⁵S]thio)triphosphate binding to membranes from humanneuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47: 848-854, 1995].

Cells are scraped from tissue culture dishes into 20 mM HEPES, 1 mMethylenediaminetetraacetic acid, then centrifuged at 500×g for 10 min.Cells are resuspended in this buffer and homogenized with a PolytronHomogenizer.

The homogenate is centrifuged at 27,000×g for 15 min., and the pelletresuspended in buffer A, containing: 20 mM HEPES, 10 mM MgCl₂, 100 mMNaCl, pH 7.4. The suspension is recentrifuged at 20,000×g and suspendedonce more in buffer A. For the binding assay, membranes (8-15 μgprotein) are incubated with [³⁵S]GTP S (50 pM), GDP (10 μM), with andwithout the TM of interest, in a total volume of 1.0 ml, for 60 min. at25° C. Samples are filtered over glass fibre filters and counted asdescribed for the binding assays.

Example 18 Preparation of a LC/A and H_(N)/A Backbone Clones

The following procedure creates the LC and H_(N) fragments for use asthe component backbone for multidomain fusion expression. This exampleis based on preparation of a serotype A based clone (SEQ ID NO:27 andSEQ ID NO:28), though the procedures and methods are equally applicableto the other serotypes [illustrated by the sequence listing for serotypeB (SEQ ID NO:29 and SEQ ID NO:30) and serotype C (SEQ ID NO:31 and SEQID NO:32)].

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector, selected dueto the lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector, which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-SalI-PstI-HindIII). Afragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of Protease (e.g. LC/A) Insert

The LC/A (SEQ ID NO:27) is created by one of two ways:

The DNA sequence is designed by back translation of the LC/A amino acidsequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. BamHI/SalI recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence, maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the LC/A open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and SalI restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a supplier (forexample MWG or Sigma-Genosys), so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg²⁺ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences which are not compatible with the cloning system are thenremoved using site directed mutagenesis [for example, using Quickchange(Stratagene Inc.)].

Preparation of Translocation (e.g. H_(N)) Insert

The H_(N)/A (SEQ ID NO:28) is created by one of two ways:

The DNA sequence is designed by back translation of the H_(N)/A aminoacid sequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO)] using one of a variety of reverse translation softwaretools [for example EditSeq best E. coli reverse translation (DNASTARInc.), or Backtranslation tool v2.0 (Entelechon)]. is A PstI restrictionsequence added to the N-terminus and XbaI-stop codon-HindIII to theC-terminus ensuring the correct reading frame is maintained. The DNAsequence is screened (using software such as MapDraw, DNASTAR Inc.) forrestriction enzyme cleavage sequences incorporated during the backtranslation. Any sequences that are found to be common to those requiredby the cloning system are removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences which are not compatible with the cloning systemare then removed using site directed mutagenesis [for example usingQuickchange (Stratagene Inc.)].

Example 19 Preparation of a LC/A-nociceptin-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the H_(N)-Chain)

Preparation of Linker-nociceptin-spacer Insert

The LC-H_(N) linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype A linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and H_(N)) is 23 amino acids long and has the sequenceVRGIITSKTKSLDKGYNKALNDL (amino acids 2-24 of SEQ ID NO: 111). Withinthis sequence, it is understood that proteolytic activation in nature isleads to an H_(N) domain that has an N-terminus of the sequence ALNDL.This sequence information is freely available from available databasesources such as GenBank (accession number P10845) or Swissprot(accession locus BXA1_CLOBO). Into this linker a Factor Xa site,nociceptin and spacer are incorporated; and using one of a variety ofreverse translation software tools [for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)], the DNA sequence encoding the linker-ligand-spacer regionis determined. Restriction sites are then incorporated into the DNAsequence and can be arranged as BamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:33). It is important to ensure the correct reading frame ismaintained for the spacer, nociceptin and restriction sequences and thatthe XbaI sequence is not preceded by the bases, TC, which would resulton DAM methylation. The DNA sequence is screened for restrictionsequence incorporation, and any additional sequences are removedmanually from the remaining sequence ensuring common E. coli codon usageis maintained. E. coli codon usage is assessed by reference to softwareprograms such as Graphical

Codon Usage Analyser (Geneart), and the % GC content and codon usageratio assessed by reference to published codon usage tables (forexample, GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Preparation of the LC/A-nociceptin-H_(N)/A Fusion

In order to create the LC-linker-nociceptin-spacer-H_(N) construct (SEQID NO:39), the pCR 4 vector encoding the linker (SEQ ID NO:33) iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of the LC/ADNA (SEQ ID NO:27) cleaved with BamHI+SalI. The resulting plasmid DNA isthen cleaved with PstI+XbaI restriction enzymes and serves as therecipient vector for the insertion and ligation of the H_(N)/A DNA (SEQID NO:28) cleaved with PstI+XbaI. The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:39) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID NO:40.

Example 20 Preparation of a Nociceptin-LC/A-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the LC-Chain)

The LC/A-H_(N)/A backbone is constructed as described in Example 19using the synthesised A serotype linker with the addition of a Factor Xasite for activation, arranged as BamHI-SalI-linker-proteasesite-linker-PstI-XbaI-stop codon-HindIII (SEQ ID NO:34). TheLC/A-H_(N)/A backbone and the synthesised N-terminal presentationnociceptin insert (SEQ ID NO:35) are cleaved with BamHI+HindIIIrestriction enzymes, gel purified and ligated together to create anociceptin-spacer-LC-linker-H_(N). The ORF (SEQ ID NO:41) is then cutout using restriction enzymes AvaI+XbaI for transfer into expressionvectors for expression to result in a fusion protein of the sequenceillustrated in SEQ ID NO:42.

Example 21 Preparation of a LC/C-nociceptin-H_(N)/C Fusion Protein

Following the methods used in Examples 1 and 2, the LC/C (SEQ ID NO:31)and H_(N)/C (SEQ ID NO:32) are created and inserted into the C serotypelinker arranged as BamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:36). The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:43) for expression as aprotein of the sequence illustrated in SEQ ID NO:44.

Example 22 Preparation of a LC/C-nociceptin-H_(N)/C Fusion Protein witha Serotype A Activation Sequence

Following the methods used in Examples 1 and 2, the LC/C (SEQ ID NO:31)and H_(N)/C (SEQ ID NO:32) are created and inserted into the A serotypelinker arranged as BamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:33). The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:45) for expression as aprotein of the sequence illustrated in SEQ ID NO:46.

Example 23 Preparation of a LC/A-met Enkephalin-H_(N)/A Fusion Protein

Due to the small, five-amino acid, size of the met-enkephalin ligand theLC/A-met enkephalin-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) as a template.Oligonucleotides are designed encoding the YGGFM met-enkephalin peptide(SEQ ID NO:112), ensuring standard E.coli codon usage is maintained andno additional restriction sites are incorporated, flanked by sequencescomplimentary to the linker region of the LC/A-nociceptin-H_(N)/A fusion(SEQ ID NO:39) either side on the nociceptin section. The SDM product ischecked by sequencing and the final construct containing theLC-linker-met enkephalin-spacer-H_(N) ORF (SEQ ID NO:47) for expressionas a protein of the sequence illustrated in SEQ ID NO:48.

Example 24 Preparation of a LC/A-13 Endorphin-H_(N)/A Fusion Protein

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID NO:27)and H_(N)/A (SEQ ID NO:28) are created and inserted into the A serotypeβ endorphin linker arranged as BamHI-SalI-linker-protease site-βendorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID NO:37).The final construct contains the LC-linker-β endorphin-spacer-H_(N) ORF(SEQ ID NO:49) for expression as a protein of the sequence illustratedin SEQ ID NO:50.

Example 25 Preparation of a LC/A-nociceptin Variant-H_(N)/A FusionProtein

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID NO:27)and H_(N)/A (SEQ ID NO:28) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-SalI-linker-proteasesite-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII(SEQ ID NO:38). The final construct contains the LC-linker-nociceptinvariant-spacer-H_(N) ORF (SEQ ID NO:51) for expression as a is proteinof the sequence illustrated in SEQ ID NO:52.

Example 26 Purification Method for LC/A-nociceptin-H_(N)/A FusionProtein

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Make the thawed cellpaste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate onice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 micronsensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4°C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO₄ chargedChelating column (20-30 ml column is sufficient) equilibrated with 50 mMHEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mMimidazol, wash away the non-specific bound protein and elute the fusionprotein with 100 mM imidazol. Dialyse the eluted fusion protein against5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measurethe OD of the dialysed fusion protein. Add 1 unit of factor Xa per 100μg fusion protein and Incubate at 25° C. static overnight. Load onto a0.1 M NiSO₄ charged Chelating column (20-30 ml column is sufficient)equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column tobaseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of10 and 40 mM imidazol, wash away the non-specific bound protein andelute the fusion protein with 100 mM imidazol. Dialyse the eluted fusionprotein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C.overnight and concentrate the fusion to about 2 mg/ml, aliquot sampleand freeze at −20° C. Test purified protein using OD, BCA, purityanalysis and SNAP-25 assessments.

Example 27 Preparation of a LC/A-nociceptin-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the H_(N)-Chain)

The linker-nociceptin-spacer insert is prepared as described in Example19.

Preparation of the LC/A-nociceptin-H_(N)/A Fusion

In order to create the LC-linker-nociceptin-spacer-H_(N) construct (SEQID NO:39), the pCR 4 vector encoding the linker (SEQ ID NO:33) iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient for insertion and ligation of the LC/A DNA (SEQID NO:27) also cleaved with BamHI+SalI. The resulting plasmid DNA isthen cleaved with BamHI+HindIII restriction enzymes and the LC/A-linkerfragment inserted into a similarly cleaved vector containing a uniquemultiple cloning site for BamHI, SalI, PstI, and HindIII such as thepMAL vector (NEB). The H_(N)/A DNA (SEQ ID NO:28) is then cleaved withPstI+HindIII restriction enzymes and inserted into the similarly cleavedpMAL-LC/A-linker construct. The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:39) for expression as aprotein of the sequence illustrated in SEQ ID NO:40.

Example 28 Preparation of a Nociceptin-LC/A-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the LC-Chain)

In order to create the nociceptin-spacer-LC/A-H_(N)/A construct, an Aserotype linker with the addition of a Factor Xa site for activation,arranged as BamHI-SalI-linker-protease site-linker-PstI-XbaI-stopcodon-HindIII (SEQ ID NO:34) is synthesised as described in Example 27.The pCR 4 vector encoding the linker is cleaved with BamHI+SalIrestriction enzymes. This cleaved vector then serves as the recipientfor insertion and ligation of the LC/A DNA (SEQ ID NO:27) also cleavedwith BamHI+SalI. The resulting plasmid DNA is then cleaved withBamHI+HindIII restriction enzymes and the LC/A-linker fragment insertedinto a similarly cleaved vector containing the synthesised N-terminalpresentation nociceptin insert (SEQ ID NO:35). This construct is thencleaved with AvaI+HindIII and inserted into an expression vector such asthe pMAL plasmid (NEB). The H_(N)/A DNA (SEQ ID NO:28) is then cleavedwith PstI+HindIII restriction enzymes and inserted into the similarlycleaved pMAL-nociceptin-LC/A-linker construct. The final constructcontains the nociceptin-spacer-LC/A-H_(N)/A ORF (SEQ ID NO:63) forexpression as a protein of the sequence illustrated in SEQ ID NO:64.

Example 29 Preparation and Purification of an LC/A-nociceptin-H_(N)/AFusion Protein Family with Variable Spacer Length

Using the same strategy as employed in Example 19, a range of DNAlinkers were prepared that encoded nociceptin and variable spacercontent. Using one of a variety of reverse translation software tools[for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)], the DNA sequence encoding thelinker-ligand-spacer region is determined. Restriction sites are thenincorporated into the DNA sequence and can be arranged asBamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:65 to SEQ ID NO:69). It is important to ensure the correct readingframe is maintained for the spacer, nociceptin and restriction sequencesand that the XbaI sequence is not preceded by the bases, TC which wouldresult on DAM methylation. The DNA sequence is screened for restrictionsequence incorporation and any additional sequences are removed manuallyfrom the remaining sequence ensuring common E. coli codon usage ismaintained. E. coli codon usage is assessed by reference to softwareprograms such as Graphical Codon Usage Analyser (Geneart), and the % GCcontent and codon usage ratio assessed by reference to published codonusage tables (for example GenBank Release 143, 13 Sep. 2004). Thisoptimised DNA sequence is then commercially synthesized (for example byEntelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4vector.

The spacers that were created included:

TABLE 2 SEQ ID NO: of the Code Protein sequence of the linker linker DNAGS10 ALAGGGGSALVLQ 113 GS15 ALAGGGGSGGGGSALVLQ 114 GS25ALAGGGGSGGGGSGGGGSGGGGSALVLQ 115 GS30 ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ116 HX27 ALAAEAAAKEAAAKEAAAKAGGGGSALVLQ 117

By way of example, in order to create the LC/A-CPN(GS15)-H_(N)/A fusionconstruct (SEQ ID NO:70), the pCR 4 vector encoding the linker (SEQ IDNO:66) is cleaved with BamHI+SalI restriction enzymes. This cleavedvector then serves as the recipient vector for insertion and ligation ofthe LC/A DNA (SEQ ID NO:27) also cleaved with BamHI+SalI. The resultingplasmid DNA is then cleaved with BamHI+HindIII restriction enzymes andthe LC/A-linker fragment inserted into a similarly cleaved vectorcontaining a unique multiple cloning site for BamHI, SalI, PstI, andHindIII such as the pMAL vector (NEB). The H_(N)/A DNA (SEQ ID NO:28) isthen cleaved with PstI+HindIII restriction enzymes and inserted into thesimilarly cleaved pMAL-LC/A-linker construct. The final constructcontains the LC/A-CPN(GS15)-H_(N)/A ORF (SEQ ID NO:70) for expression asa protein of the sequence illustrated in SEQ ID NO:71.

As a further example, to create the LC/A-CPN(GS25)-H_(N)/A fusionconstruct (SEQ ID NO:72), the pCR 4 vector encoding the linker (SEQ IDNO:67) is cleaved with BamHI+SalI restriction enzymes. This cleavedvector then serves as the recipient vector for insertion and ligation ofthe LC/A DNA (SEQ ID NO:27) cleaved with BamHI+SalI. The resultingplasmid DNA is then cleaved with BamHI+HindIII restriction enzymes andthe LC/A-linker fragment inserted into a similarly cleaved vectorcontaining a unique multiple cloning site for BamHI, SalI, PstI, andHindIII such as the pMAL vector (NEB). The H_(N)/A DNA (SEQ ID NO:28) isthen cleaved with PstI+HindIII restriction enzymes and inserted into thesimilarly cleaved pMAL-LC/A-linker construct. The final constructcontains the LC/A-CPN(GS25)-H_(N)/A ORF (SEQ ID NO:72) for expression asa protein of the sequence illustrated in SEQ ID NO:73.

Variants of the LC/A-CPN-H_(N)/A fusion consisting of GS10, GS30 andHX27 are similarly created. Using the purification methodology describedin Example 26, fusion protein is purified from E. coli cell paste. FIG.12 illustrates the purified product obtained in the case ofLC/A-CPN(GS10)-H_(N)/A, LC/A-CPN(GS15)-H_(N)/A, LC/A-CPN(GS25)-H_(N)/A,LC/A-CPN(GS30)-H_(N)/A and LC/A-CPN(HX27)-H_(N)/A.

Example 30 Assessment of in vitro Efficacy of an LC/A-nociceptin-H_(N)/AFusion

Fusion protein prepared according to Examples 2 and 9 was assessed inthe eDRG neuronal cell model.

Assays for the inhibition of substance P release and cleavage of SNAP-25have been previously reported (Duggan et al., 2002, J. Biol. Chem., 277,34846-34852). Briefly, dorsal root ganglia neurons are harvested from15-day-old fetal Sprague-Dawley rats and dissociated cells plated onto24-well plates coated with Matrigel at a density of 1×10⁶ cells/well.One day post-plating the cells are treated with 10 μM cytosineβ-D-arabinofuranoside for 48 h. Cells are maintained in Dulbecco'sminimal essential medium supplemented with 5% heat-inactivated fetalbovine serum, 5 mM L-glutamine, 0.6% D-glucose, 2% B27 supplement, and100 ng/ml 2.5S mouse nerve growth factor. Cultures are maintained for 2weeks at 37° C. in 95% air/5% CO₂ before addition of test materials.

Release of substance P from eDRG is assessed by enzyme-linkedimmunosorbent assay. Briefly, eDRG cells are washed twice with lowpotassium-balanced salt solution (BSS: 5 mM KCl, 137 mM NaCl, 1.2 mMMgCl₂, 5 mM glucose, 0.44 mM KH₂PO₄, 20 mM HEPES, pH 7.4, 2 mM CaCl₂).Basal samples are obtained by incubating each well for 5 min. with 1 mlof low potassium BSS. After removal of this buffer, the cells arestimulated to release by incubation with 1 ml of high potassium buffer(BSS as above with modification to include 100 mM KCl isotonicallybalanced with NaCl) for 5 min. All samples are removed to tubes on iceprior to assay of substance P. Total cell lysates are prepared byaddition of 250 μl of 2 M acetic acid/0.1% trifluoroacetic acid to lysethe cells, centrifugal evaporation, and resuspension in 500 μl of assaybuffer. Diluted samples are assessed for substance P content. SubstanceP immunoreactivity is measured using Substance P Enzyme Immunoassay Kits(Cayman Chemical Company or R&D Systems) according to manufacturers'instructions. Substance P is expressed in pg/ml relative to a standardsubstance P curve run in parallel.

SDS-PAGE and Western blot analysis were performed using standardprotocols (Novex). SNAP-25 proteins were resolved on a 12% Tris/glycinepolyacrylamide gel (Novex) and subsequently transferred tonitrocellulose membrane. The membranes were probed with a monoclonalantibody (SMI-81) that recognises cleaved and intact SNAP-25. Specificbinding was visualised using peroxidase-conjugated secondary antibodiesand a chemiluminescent detection system. Cleavage of SNAP-25 wasquantified by scanning densitometry (Molecular Dynamics Personal SI,ImageQuant data analysis software). Percent SNAP-25 cleavage wascalculated according to the formula: (Cleaved SNAP-25/(Cleaved+IntactSNAP-25))×100.

Following exposure of eDRG neurons to an LC/A-nociceptin-H_(N)/A fusion(termed CPN-A), both inhibition of substance P release and cleavage ofSNAP-25 are observed (FIG. 13). After 24 h exposure to the fusion, 50%of maximal SNAP-25 cleavage is achieved by a fusion concentration of6.3±2.5 nM.

The effect of the fusion is also assessed at defined time pointsfollowing a 16 h exposure of eDRG to CPN-A. FIG. 14 illustrates theprolonged duration of action of the CPN-A fusion protein, withmeasurable activity still being observed at 28 days post exposure.

Example 31 Assessment of in vitro Efficacy of an LC/A-nociceptinVariant-H_(N)/A Fusion

Fusion protein prepared according to Examples 8 and 9 was assessed inthe eDRG neuronal cell mode using the method described in Example 30.

Following exposure of eDRG neurons to an LC/A-nociceptin variant-H_(N)/Afusion (termed CPNv-A), both inhibition of substance P release andcleavage of SNAP-25 are observed. After 24 h exposure to the fusion, 50%of maximal SNAP-25 cleavage is achieved by a fusion concentration of1.4±0.4 nM (FIG. 15).

The effect of the fusion is also assessed at defined time pointsfollowing a 16 h exposure of eDRG to CPN-A. FIG. 16 illustrates theprolonged duration of action of the CPN-A fusion protein, withmeasurable activity still being observed at 24 days post exposure.

The binding capability of the CPNv-A fusion protein is also assessed incomparison to the CPN-A fusion. FIG. 17 illustrates the results of acompetition experiment to determine binding efficacy at the ORL-1receptor. CPNv-A is demonstrated to displace [3H]-nociceptin, therebyconfirming that access to the receptor is possible with the ligand inthe central presentation format.

Example 32 Preparation of an LC/A-nociceptin Variant-H_(N)/A FusionProtein that is Activated by Treatment with Enterokinase

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID NO:27)and H_(N)/A (SEQ ID NO:28) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-SalI-linker-enterokinaseprotease site-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID NO:74). The final construct contains theLC-linker-nociceptin variant-spacer-H_(N) ORF sequences (SEQ ID NO:75)for expression as a protein of the sequence illustrated in SEQ ID NO:76.The fusion protein is termed CPNv(Ek)-A. FIG. 18 illustrates thepurification of CPNv(Ek)-A from E. coli following the methods used inExample 26 but using Enterokinase for activation at 0.00064 μg per 100μg of fusion protein.

Example 33 Assessment of in vitro Efficacy of an LC/A-nociceptinVariant-H_(N)/A Fusion that has Been Activated by Treatment withEnterokinase

The CPNv(Ek)-A prepared in Example 32 is obtained in a purified form andapplied is to the eDRG cell model to assess cleavage of SNAP-25 (usingmethodology from Example 30). FIG. 19 illustrates the cleavage ofSNAP-25 following 24 h exposure of eDRG to CPNv(Ek)-A. The efficiency ofcleavage is observed to be similar to that achieved with the FactorXa-cleaved material, as recorded in Example 31.

Example 34 Preparation of an LC/C-nociceptin Variant-H_(N)/C FusionProtein with a Factor Xa Activation Linker Derived from Serotype A

Following the methods used in Example 21, the LC/C (SEQ ID NO:31) andH_(N)/C (SEQ ID NO:32) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-SalI-linker-nociceptinvariant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID NO:77).The final construct contains the LC-linker-nociceptinvariant-spacer-H_(N) ORF sequences (SEQ ID NO:78) for expression as aprotein of the sequence illustrated in SEQ ID NO:79. The fusion proteinis termed CPNv-C (act. A). FIG. 20 illustrates the purification ofCPNv-C (act. A) from E. coli following the methods used in Example 26.

Example 35 Assessment of in vitro Efficacy of an LC/C-nociceptinVariant-H_(N)/C Fusion Protein

Following the methods used in Example 26, the CPNv-C (act. A) preparedin Example 34 is obtained in a purified form and applied to the eDRGcell model to assess cleavage of SNAP-25 (using methodology from Example30). After 24 h exposure to the fusion, 50% of maximal syntaxin cleavageis achieved by a fusion concentration of 3.1±2.0 nM. FIG. 21 illustratesthe cleavage of syntaxin following 24 h exposure of eDRG to CPNv-C (act.A).

Example 36 Assessment of in vivo Efficacy of an LC/A-nociceptin-HN/AFusion

The ability of an LC/A-nociceptin-H_(N)/A fusion (CPN/A) to inhibitacute capsaicin-induced mechanical allodynia is evaluated followingsubcutaneous intraplantar is injection in the rat hind paw. Test animalsare evaluated for paw withdrawal frequency (PWF %) in response to a 10 gVon Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study, after subcutaneous treatment with CPN/A butbefore capsaicin, and following capsaicin challenge post-injection ofCPN/A (average of responses at 15′ and 30′). Capsaicin challenge isachieved by injection of 10 μL of a 0.3% solution. Sample dilutions areprepared in 0.5% BSA/saline. FIG. 22 illustrates the reversal ofmechanical allodynia that is achieved by pre-treatment of the animalswith a range of concentrations of LC/A-nociceptin-HN/A fusion.

The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibitstreptozotocin (STZ)-induced mechanical (tactile) allodynia in rats isevaluated. STZ-induced mechanical allodynia in rats is achieved byinjection of streptozotocin (i.p. or i.v.) which yields destruction ofpancreatic β-cells leading to loss of insulin production, withconcomitant metabolic stress (hyperglycemia and hyperlipidemia). Assuch, STZ induces Type I diabetes. In addition, STZ treatment leads toprogressive development of neuropathy, which serves as a model ofchronic pain with hyperalgesia and allodynia that may reflect signsobserved in diabetic humans (peripheral diabetic neuropathy).

Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ incitrate buffer (I.V.) and blood glucose and lipid are measured weekly todefine the readiness of the model. Paw Withdrawal Threshold (PWT) ismeasured in response to a Von Frey filament stimulus series over aperiod of time. Allodynia is said to be established when the PWT on twoconsecutive test dates (separated by 1 week) measures below 6 g on thescale. At this point, rats are randomized to either a saline group(negative efficacy control), gabapentin group (positive efficacycontrol) or a test group (CPN/A). Test materials (20-25 μl) are injectedsubcutaneously as a single injection (except gabapentin) and the PWT ismeasured at 1 day post-treatment and periodically thereafter over a2-week period. Gabapentin (30 mg/kg i.p. @3 ml/kg injection volume) isinjected daily, 2 hours prior to the start of PWT testing. FIG. 23illustrates the reversal of allodynia achieved by pre-treatment of isthe animals with 750 ng of CPN/A. Data were obtained over a 2-weekperiod after a single injection of CPN/A

Example 37 Assessment of in vivo Efficacy of an LC/A-nociceptinVariant-H_(N)/A Fusion

The ability of an LC/A-nociceptin variant-H_(N)/A fusion (CPNv/A) toinhibit capsaicin-induced mechanical allodynia is evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalsare evaluated for paw withdrawal frequency (PWF %) in response to a 10 gVon Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat); after subcutaneous intraplantartreatment with CPNv/A but before capsaicin (Pre-CAP); and followingcapsaicin challenge post-injection of CPNv/A (average of responses at15′ and 30′; CAP). Capsaicin challenge is achieved by injection of 10 μLof a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline.

FIG. 24 illustrates the reversal of allodynia that is achieved bypre-treatment of the animals with a range of concentrations ofLC/A-nociceptin variant-H_(N)/A fusion in comparison to the reversalachieved with the addition of LC/A-nociceptin-H_(N)/A fusion. These dataare expressed as a normalized paw withdrawal frequency differential, inwhich the difference between the peak response (post-capsaicin) and thebaseline response (pre-capsaicin) is expressed as a percentage. Withthis s analysis, it can be seen that CPNv/A is more potent than CPN/Asince a lower dose of CPNv/A is required to achieve similar analgesiceffect to that seen with CPN/A.

Example 38 Preparation of an LC/A-Leu Enkephalin-H_(N)/A Fusion Protein

Due to the small, five-amino acid, size of the leu-enkephalin ligand theLC/A-leu enkephalin-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) as a template.Oligonucleotides are designed encoding the YGGFL leu-enkephalin peptide,ensuring standard E. coli codon usage is maintained and no additionalrestriction sites are incorporated, flanked by sequences complimentaryto the linker region of the LC/A-nociceptin-H_(N)/A fusion (SEQ IDNO:39) either side on the nociceptin section. The SDM product is checkedby sequencing and the final construct containing the LC-linker-leuenkephalin-spacer-H_(N) ORF (SEQ ID NO:80) for expression as a proteinof the sequence illustrated in SEQ ID NO:81. The fusion protein istermed CPLE-A. FIG. 25 illustrates the purification of CPLE-A from E.coli following the methods used in Example 26.

Example 39 Expression and Purification of an LC/A-Beta-Endorphin-H_(N)/AFusion Protein

Following the methods used in Example 26, and with theLC/A-beta-endorphin-H_(N)/A fusion protein (termed CPBE-A) created inExample 24, the CPBE-A is purified from E. coli. FIG. 26 illustrates thepurified protein as analysed by SDS-PAGE.

Example 40 Preparation of an LC/A-nociceptin Mutant-H_(N)/A FusionProtein

Due to the single amino acid modification necessary to mutate thenociceptin sequence at position 1 from a Phe to a Tyr, theLC/A-nociceptin mutant-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) as a template.Oligonucleotides are designed encoding tyrosine at position 1 of thenociceptin s sequence, ensuring standard E. coli codon usage ismaintained and no additional restriction sites are incorporated, flankedby sequences complimentary to the linker region of theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) either side on thenociceptin section. The SDM product is checked by sequencing and thefinal construct containing the LC/A-nociceptin mutant-spacer-H_(N)/Afusion ORF (SEQ ID NO:82) for expression as a protein of the sequenceillustrated in SEQ ID NO:83. The fusion protein is termed CPOP-A. FIG.27 illustrates the purification of CPOP-A from E. coli following themethods used in Example 26.

Example 41 Preparation and Assessment of an LC/A-nociceptin Variant isMutant-H_(N)/A Fusion Protein

Due to the single amino acid modification necessary to mutate thenociceptin sequence at position 1 from a Phe to a Tyr, theLC/A-nociceptin variant mutant-H_(N)/A fusion is created by sitedirected mutagenesis [for example using Quickchange (Stratagene Inc.)]using the LC/A-nociceptin variant-H_(N)/A fusion (SEQ ID NO:51) as atemplate. Oligonucleotides are designed encoding tyrosine at position 1of the nociceptin sequence, ensuring standard E. coli codon usage ismaintained and no additional restriction sites are incorporated, flankedby sequences complimentary to the linker region of the LC/A-nociceptinvariant-H_(N)/A fusion (SEQ ID NO:51) either side on the nociceptinsection. The SDM product is checked by sequencing and the finalconstruct containing the LC/A-nociceptin mutant-spacer-H_(N)/A fusionORF (SEQ ID NO:84) for expression as a protein of the sequenceillustrated in SEQ ID NO:85. The fusion protein is termed CPOPv-A. FIG.28 illustrates the purification of CPOPv-A from E. coli following themethods used in Example 26.

Using methodology described in Example 30, CPOPv-A is assessed for itsability to cleave SNAP-25 in the eDRG cell model. FIG. 29 illustratesthat CPOPv-A is able to cleave SNAP-25 in the eDRG model, achievingcleavage of 50% of the maximal SNAP-25 after exposure of the cells toapproximately 5.9 nM fusion for 24 h.

Example 42 Preparation of an IgA Protease-nociceptin Variant-H_(N)/AFusion Protein

The IgA protease amino acid sequence was obtained from freely availabledatabase sources such as GenBank (accession number P09790). Informationregarding the structure of the N. Gonorrhoeae IgA protease gene isavailable in the literature (Pohlner et al., Gene structure andextracellular secretion of Neisseria gonorrhoeae IgA protease, Nature,1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon),the DNA sequence encoding the IgA protease modified for E. coliexpression was determined. A BamHI recognition sequence was incorporatedat the 5′ end and a codon encoding a cysteine amino acid and SalIrecognition sequence is were incorporated at the 3′ end of the IgA DNA.The DNA sequence was screened using MapDraw, (DNASTAR Inc.) forrestriction enzyme cleavage sequences incorporated during the backtranslation. Any cleavage sequences that are found to be common to thoserequired for cloning were removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage was assessed Graphical Codon Usage Analyser (Geneart), andthe % GC content and codon usage ratio assessed by reference topublished codon usage tables. This optimised DNA sequence (SEQ ID NO:86)containing the IgA open reading frame (ORF) is then commerciallysynthesized.

The IgA (SEQ ID NO:86) is inserted into the LC-linker-nociceptinvariant-spacer-H_(N) ORF (SEQ ID NO:51) using BamHI and SalI restrictionenzymes to replace the LC with the IgA protease DNA. The final constructcontains the IgA-linker-nociceptin variant-spacer-H_(N) ORF (SEQ IDNO:87) for expression as a protein of the sequence illustrated in SEQ IDNO:88.

Example 43 Preparation and Assessment of a Nociceptin TargetedEndopeptidase Fusion Protein with a Removable Histidine Purification Tag

DNA was prepared that encoded a Factor Xa removable his-tag (his6),although it is clear that alternative proteases site such asEnterokinase and alternative purification tags such as longer histidinetags are also possible. Using one of a variety of reverse translationsoftware tools [for example EditSeq best E. coli reverse translation(DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNAsequence encoding the Factor Xa removable his-tag region is determined.Restriction sites are then incorporated into the DNA sequence and can bearranged as NheI-linker-SpeI-PstI-H_(N)/A-XbaI-LEIEGRSGHHHHHHStopcodon-HindIII (SEQ ID NO:89). The DNA sequence is screened forrestriction sequence incorporated and any additional sequences areremoved manually from the remaining sequence ensuring common E. colicodon usage is maintained. E. coli codon usage is assessed by referenceto software programs such as Graphical Codon Usage Analyser (Geneart),and the % GC content and codon usage ratio assessed by is reference topublished codon usage tables (for example GenBank Release 143, 13 Sep.2004). This optimised DNA sequence is then commercially synthesized (forexample by Entelechon, Geneart or Sigma-Genosys) and is provided in thepCR 4 vector. In order to create CPNv-A-FXa-HT (SEQ ID NO:90, removablehis-tag construct) the pCR 4 vector encoding the removable his-tag iscleaved with NheI and HindIII. The NheI-HindIII fragment is theninserted into the LC/A-CPNv-H_(N)/A vector (SEQ ID NO:51) that has alsobeen cleaved by NheI and HindIII. The final construct contains theLC/A-linker-nociceptin variant-spacer-H_(N)-FXa-Histag-HindIII ORFsequences (SEQ ID NO:90) for expression as a protein of the sequenceillustrated in SEQ ID NO:91. FIG. 30 illustrates the purification ofCPNv-A-FXa-HT from E. coli following the methods used in Example 26.

Example 44 Preparation of a Leu-enkephalin Targeted Endopeptidase FusionProtein Containing a Translocation Domain Derived from Diphtheria Toxin

The DNA sequence is designed by back translation of the amino acidsequence of the translocation domain of the diphtheria toxin (obtainedfrom freely available database sources such as GenBank (accession number1XDTT) using one of a variety of reverse translation software tools [forexample EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. Restriction sites are thenincorporated into the DNA sequence and can be arranged asNheI-Linker-SpeI-PstI-diphtheria translocation domain-XbaI-stopcodon-HindIII (SEQ ID NO:92). PstI/XbaI recognition sequences areincorporated at the 5′ and 3′ ends of the translocation domainrespectively of the sequence maintaining the correct reading frame. TheDNA sequence is screened (using software such as MapDraw, DNASTAR Inc.)for restriction enzyme cleavage sequences incorporated during the backtranslation. Any cleavage sequences that are found to be common to thoserequired by the cloning system are removed manually from the proposedcoding sequence ensuring common E. coli codon usage is maintained. E.coli codon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the diphtheria translocation domain is then commerciallysynthesized as NheI-Linker-SpeI-PstI-diphtheria translocationdomain-XbaI-stop codon-HindIII (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector (Invitrogen). The pCR4 vector encoding the diphtheria translocation domain is cleaved withNhel and XbaI. The NheI-XbaI fragment is then inserted into theLC/A-CPLE-H_(N)/A vector (SEQ ID NO:80) that has also been cleaved byNheI and XbaI. The final construct contains theLC/A-leu-enkephalin-spacer-diphtheria translocation domain ORF sequences(SEQ ID NO:93) for expression as a protein of the sequence illustratedin SEQ ID NO:94.

Example 45 Preparation of a Nociceptin Variant Targeted EndopeptidaseFusion Protein Containing a LC Domain Derived from Tetanus Toxin

The DNA sequence is designed by back translation of the tetanus toxin LCamino acid sequence (obtained from freely available database sourcessuch as GenBank (accession number X04436) using one of a variety ofreverse translation software tools [for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)]. BamHI/SalI recognition sequences are incorporated at the5′ and 3′ ends respectively of the sequence maintaining the correctreading frame (SEQ ID NO:95). The DNA sequence is screened (usingsoftware such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavagesequences incorporated during the back translation. Any cleavagesequences that are found to be common to those required by the cloningsystem are removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage isassessed by reference to software programs such as Graphical Codon UsageAnalyser (Geneart), and the % GC content and codon usage ratio assessedby reference to published codon usage tables (for example GenBankRelease 143, 13 Sep. 2004). This optimised DNA sequence containing thetetanus toxin LC open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding theis TeNT LC is cleaved with BamHI and SalI. The BamHI-SalI fragment isthen inserted into the LC/A-CPNv-H_(N)/A vector (SEQ ID NO:51) that hasalso been cleaved by BamHI and SalI. The final construct contains theTeNT LC-linker-nociceptin variant-spacer-H_(N) ORF sequences (SEQ IDNO:96) for expression as a protein of the sequence illustrated in SEQ IDNO:97.

Example 46 Preparation of an LC/C-nociceptin Variant-H_(N)/C FusionProtein with a Native Serotype C Linker that is Susceptible to Factor XaCleavage

Following the methods used in Example 21, the LC/C (SEQ ID NO:31) andH_(N)/C (SEQ ID NO:32) are created and inserted into the C serotypenociceptin variant linker arranged as BamHI-SalI-linker-nociceptinvariant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID NO:98).The final construct contains the LC-linker-nociceptinvariant-spacer-H_(N) ORF sequences (SEQ ID NO:99) for expression as aprotein of the sequence illustrated in SEQ ID NO:100. The fusion proteinis termed CPNv-C (act. C).

The invention claimed is:
 1. A non-cytotoxic protein conjugate forinhibition or reduction of exocytic fusion in a nociceptive sensoryafferent cell, comprising: (i) a Targeting Moiety (TM), wherein said TMis a nociceptin peptide that binds to a receptor present on saidnociceptive sensory afferent cell, wherein said receptor undergoesendocytosis to be incorporated into an endosome within the nociceptivesensory afferent cell, wherein the receptor is an opioid receptor-like 1(ORL1) receptor, and wherein the TM is SEQ ID NO:6 or 8; (ii) anon-cytotoxic protease or a fragment thereof, wherein the protease orprotease fragment is a clostridial protease or a neisserial IgA proteaseor a fragment thereof, and wherein the protease or protease fragmentcleaves a protein of the exocytic fusion apparatus of said nociceptivesensory afferent cell; and (iii) a Translocation Domain, wherein theTranslocation Domain translocates the protease or protease fragment fromwithin the endosome, across the endosomal membrane, and into the cytosolof the nociceptive sensory afferent cell, and wherein the TranslocationDomain is a translocation domain of a clostridial neurotoxin or from abacterial or viral source.
 2. The non-cytotoxic conjugate of claim 1,wherein the Translocation Domain is obtained from a clostridial source.3. The non-cytotoxic conjugate of claim 2, wherein the TranslocationDomain is a botulinum H_(N) domain.
 4. The non-cytotoxic conjugate ofclaim 1, wherein the nociceptive sensory afferent cell is a primarynociceptive sensory afferent cell.
 5. The non-cytotoxic conjugateaccording to claim 1, wherein said conjugate comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:18, 56 and 58.6. The non-cytotoxic conjugate of claim 1, wherein the TM, theTranslocation Domain and the protease or fragment thereof are covalentlylinked.
 7. A pharmaceutical composition, comprising a conjugateaccording to claim 1 and a pharmaceutically acceptable carrier.
 8. Anexpression vector comprising a polynucleotide sequence encoding theconjugate of claim
 1. 9. The expression vector according to claim 8,wherein the vector comprises a polynucleotide sequence selected from thegroup consisting of SEQ ID NOs:17, 55and
 57. 10. A method of preparing aconjugate, comprising expressing the polynucleotide sequence of claim 8in a host cell.
 11. A method for treating pain in a subject, comprisingadministering to a subject a therapeutically effective amount of aconjugate of claim 1, thereby treating pain in the subject.
 12. A methodfor treating pain in a subject, comprising administering to a subject atherapeutically effective amount of a pharmaceutical compositionaccording to claim 7, thereby treating pain in the subject.